Methods and devices for harvesting and processing connective tissue precursor cells from autologous fat

ABSTRACT

Methods and devices are disclosed for processing stromal precursor cells (i.e., cells which can differentiate into connective tissue cells, such as in muscles, ligaments, or tendons) which can be obtained from fatty tissue extracts obtained via liposuction. Normal processing of a liposuction extract involves centrifugation, to concentrate the stromal cells into a semi-concentrated form called “spun fat”. That “spun fat” can then be treated by mechanical processing (such as pressure-driven extrusion through 0.5 mm holes) under conditions which can gently pry the stromal cells away from extra-cellular collagen fibers and other debris in the “spun fat”. The extruded mixture is then centrifuged again, to separate a highly-enriched population of stromal cells which is suited for injection back into the patient (along with platelet cells, if desired, to further promote tissue repair or regeneration).

BACKGROUND

This invention is in the field of medicine, surgery, and veterinarymedicine. It relates to devices and methods for transplanting cellsobtained from fatty tissue, in one region of a patient's (or animal's)body, into a different location in the same person's (or animal's) body,for purposes such as: (i) repairing damaged connective tissue, inlocations such as joints, muscles, tendons, ligaments, etc; and, (ii)cosmetic and other appearance-related surgery, such as scar revisions,repair of congenital defects, and surface enhancements.

When used to refer to surgical procedures, the terms “autologoustransplantation” and “auto-transplantation” are used interchangeably.The term “autologous” indicates that living cells are removed,extracted, or otherwise obtained from one portion of a person's body,and are subsequently implanted, transplanted, injected, or otherwiseemplaced in a different location in the body of that same person. Exceptin rare cases that do not require attention here (involving autoimmunedisorders and the like), autologous cells (defined to include cellsobtained from the same animal or human body that will be receiving thetransplanted cells) do not create a risk of rejection by the patient'sbody.

In some situations, autologous transplanted tissues remain in cohesiveform. Examples include blood vessel grafts, skin grafts, etc. Thosetypes of surgery are not relevant herein.

In the surgical procedures of interest herein, cells will be extractedin a liquefied form, from fatty tissue within a patient's body, usingmethods that fall within the medical term “liposuction”. The term“lipo-” refers to fat or fatty; accordingly, “liposuction” is usedbroadly herein, to refer to and include any type of procedures whichuses suction (via a hollow needle, cannula, or other tube) to removefat-containing tissue from the body of a human or other mammal. Sincesuction of tissue through a needle, cannula, or other tube is involved,the tissue which passes through the tube will necessarily be in sometype of a liquefied form, and “liquefied” is used broadly herein, toinclude thick and viscous cell suspensions that might also be referredto as a paste, slurry, sludge, gel, or similar terms.

The phrase, “autologous fat grafting” (abbreviated as AFG) is often usedin the medical literature, to refer to the types of procedures describedherein. However, that phrase is not used herein, since it is potentiallymisleading, since “fat” will not be transplanted back into the patient.Instead, the desired and targeted cells that are useful for these typesof procedures are a specialized class of cells, referred to hereininterchangeably as either “stromal precursor cells” or “connectivetissue precursor cells”. Those are described in some detail, below. Bymeans of certain types of processing steps described below, those cellsare extracted from the liquefied fatty tissue that is obtained vialiposuction, and extracellular fat (which initially accompanies thosedesired cells, in the liquefied tissue obtained via liposuction)preferably should be removed and discarded.

References herein to surgery (and to “surgical” procedures, operations,and the like) refer to and include medical interventions that involvephysical manipulation of tissue (as distinct from, for example,diagnosing a condition and prescribing a drug to treat the condition).For the purposes of discussion herein, liposuction is deemed to be atype of surgery, and the medical interventions described herein aredeemed to be “surgical” interventions. In the US and elsewhere, thesetypes of procedures can be performed, lawfully, only by properly trainedand licensed physicians; however, there are multiple thousands ofphysicians, in the US, who have the skills and ability to perform theseprocedures. Accordingly, even though substantial attention is devotedherein to the steps and devices that are used to carry out theprocedures described herein, it should be recognized and understood thatthe level of “ordinary skill in the art” in this particular fieldincludes physicians who already know how to perform liposuction, and whohave done it multiple times, on multiple patients. Accordingly, thediscussion herein of the steps, methods, devices, and equipment that areinvolved in liposuction must be regarded and understood as being meresummaries and overviews which are written for laymen, rather than as aninstruction manual for physicians.

With regard to whether the term “surgery” is used appropriately todescribe these procedures, it should be noted and understood that theseprocedures fall into a gray area, at the outer boundaries of “surgery”.A large number of medical procedures and interventions involveborderline areas, where it is not clear whether they fall within eitherclassic or contemporary definitions of “surgery”. Indeed, an entirespecialized branch of medicine has evolved during the past 20 years,which is commonly referred to as “sports medicine”. Most of theprocedures that are performed by “sports medicine” specialists involverepairs to connective tissues, which includes muscles, tendons, andligaments. Physicians who specialize in this branch of medicinefrequently perform procedures that fall within the classical definitionof “surgery” (i.e., they involve the physical manipulation andalteration of living tissues, which passes beyond merely handlingfluids, such as withdrawing blood). As part of that work, specialists in“sports medicine” frequently use needles, cannulas, catheters, and otherminimally-invasive tools, to manipulate tissue. However, they usually donot refer to themselves as “surgeons”, and they generally avoid the useof scalpels, incisions, or the types of surgery carried out by classical“surgeons” as that term is normally understood by laymen.

Accordingly, for the purposes of this invention, liposuction is deemedto be a form of “surgery”, since it involves the physical manipulationof tissue, and therefore falls within the classic definition of theterm. However, it should be recognized that not everyone refers to it as“surgery”, and “sports medicine” specialists (who likely will be amongthe main practitioners of the methods described herein) usually do notrefer to themselves as surgeons.

In addition, as will be recognized by veterinarians, the methods,devices, and cell preparations described herein can also be adapted foruse in veterinary medicine, such as to treat pets, or livestock. Forexample, since numerous breeds of dogs suffer from congenital hipproblems, a dog which is displaying symptoms of hip problems ordiscomfort can be injected with a cell preparation as described herein,into the hip area which is believed to be causing the problem, in thehope that such treatment will benefit the dog, and relieve or at leastreduce its pain, in the same way that similar injections into knee orhips joints can benefit human patients suffering from hip or kneeproblems. For brevity and convenience, animal treatments will not bementioned again, herein; however, any references to humans and/orpatients should be regarded as being also adaptable, by skilledveterinarians, to pet and/or livestock animals; and, following a normaland conventional practice, the term “patient” includes animals which areveterinary patients.

Returning to the subject of autologous transplantation of connectivetissue cells, fatty tissue (also known as adipose tissue) is readilyavailable in any human who is not exceptionally slender. It containslarge numbers of cells which fall within a category referred to herein,interchangeably, as either “precursor” or “progenitor” cells. Both ofthose two terms indicate that these cells have reached a stage ofpartial differentiation, and maturation. At that stage of development,they are able to complete a maturation process which will convert theminto any of several different types of fully differentiated “adult”cells.

Because these matters are crucial to understanding this invention, andbecause certain terms that are important herein have taken on variousimplications and subtleties that sometimes vary and diverge, when usedin the medical literature and the “popular press”, a digression isrequired to address some of the terms used herein.

“Stem Cells” Versus “Precursor” or “Progenitor” Cells

Because of various factors that are involved in public, political, andlegal battles over abortion and cloning, which are highly polarized anddivisive areas, the term “stem cells” has taken on very different andeven conflicting meanings, depending on who is using the term. Amongreporters, the press, and the general public, “stem cells” impliesand/or means the types of “embryonic” or “totipotent” cells which have afull, complete, and unlimited ability to develop into a complete adult.Accordingly, under the “general public and mass media” definition andinterpretation, the term “stem cell” is used only to refer to cellswhich have the potential to differentiate into ANY type of cell that isfound in a fully matured and differentiated adult.

However, in the scientific and medical literature, a very different setof meanings and implications arise. Under this definition, any cell thathas not yet fully differentiated into an “irrevocably committed andfully differentiated” adult cell will properly fall within the term,“stem cell”. Stated in other words, any cell which still has a potentialto differentiate into either of at least two (or more) different typesof adult cells, is called a “stem cell”. This is similar to the way any“stem” in nearly any type of a plant can have multiple leaves emergefrom it.

The medical definition arose, after developmental biologists discoveredand realized that “stem cells” pass through a series of differentstages, involving partial levels or degrees of what can be called“differentiation” or “commitment”. At the very earliest stage ofembryonic development, immediately after a sperm cell and an egg cellhave combined, the resulting cell (and the cells which arise during thefirst few cycles of replication) are called “totipotent” or “omnipotent”stem cells, because they can become ANY type of cell found in an adult.However, that stage is very brief, and it lasts only until a fertilizedegg has divided into about 4, 8, or 16 cells, depending on the species.

When stem cells pass beyond that early and brief “totipotent” or“omnipotent” stage (which lasts for only a few cell divisions), most ofthe cells which are found in a fetus become “multipotent” (also called“pluripotent”) stem cells. At that stage of maturation and “commitment”,they can still mature into numerous different types of cells; however,the pathways they can follow begin to become constrained, in variousways, and they can no longer form every cell type that exists in anadult animal of that species.

For example, during the early development of a human or other mammalianembryo, some cells will move into a segment of developing tissue whichwill become the liver. For at least some period of time, these earlyprogenitor cells will have the ability to become any type of liver cell,and there are numerous different types of liver cells. However, undernatural conditions that exist within an embryo, an embryonic stem cellwhich has committed to becoming a liver cell will not be able to backup, move over to a different development pathway, and become a heartcell, a brain cell, or any other type of non-liver cell.

Throughout all stages of infancy, adolescence, and adulthood, everyinternal organ contains numerous “multipotent” or “pluripotent” stemcells. In any particular organ, these types of cells will retain theability to complete a process of differentiation and maturation, in amanner which can create a variety of different types of new cells, whichcan replace aging cells that can no longer fully perform their cellularfunctions. This is an absolutely crucial function, which is described inmore detail below, and it helps explain the surprisingly large number ofsuch cells, in fatty tissue. Under the medical definition, these typesof “multipotent” or “pluripotent” precursor cells are labeled as “stemcells”. However, that use of the term directly conflicts with the“general public and mass media” definition of “stem cells”.

Accordingly, to avoid “unwanted baggage, and unintended meanings” thatcan arise when “stem cells” are being discussed, that term is notpreferred herein, and terms that are more scientific and less divisiveare used, such as “progenitor” or “precursor” cells.

Two additional terms requires attention; those terms are “stroma” and“stromal”. In medical terminology, the term “stroma” refers to thebiomechanical framework (or scaffolding, matrix, support, or similarterms) of an internal organ, muscle, or similar non-bony tissue. Thecorresponding adjective is “stromal”. Accordingly, stromal cells includecells which contribute to the strength, cohesiveness, flexibility,elasticity, integrity, and other structural and biomechanical traits ofan organ, muscle, tendon, ligament, or other type of connective tissue.Since the types of stromal tissue that is of interest herein is alsoreferred to as “connective tissue”, the term “stromal precursor cells”is used interchangeably with “connective tissue precursor cells”.

Briefly, “stromal precursor cells” includes cells which still have anability to differentiate and mature into any of several different typesof connective tissues, which can include muscles, tendons, ligaments,etc. Accordingly, these types of precursor cells are of great interestto “sports medicine” specialists, and to any physicians who are facedwith the task of repairing, reconstructing, or supplementing varioustypes of connective tissues that have become injured, infected,chronically sore, or otherwise damaged, or which suffer from congenitaldefects, problems that have arisen due to aging or senescence, etc. Forconvenience, connective tissue which is suffering from any of thosetypes of conditions, at a level severe enough to require or meritmedical intervention as described herein, is referred to herein as“damaged” tissue.

Types and Examples of Connective Tissue Repairs

Because of various aspects of human physiology and activity (which leadto different types of stresses being imposed on different joints), themost common types of connective tissue injuries or problems involvecertain specific joints, including the following:

1. rotator cuff problems, problems involving certain other muscles,tendons, and/or ligaments, in shoulder joints;

2. hip and/or groin problems, involving either “hyaline” cartilage(i.e., the type of smooth-surfaced cartilage which coats a bone surfacein an articulating joint), or various types of strains, tears, or otherinjuries to muscles, tendons, or ligaments in the region of the pelvisand upper thighs;

3. knee problems, which can include cartilage, ligament, tendon, ormuscle problems;

4. ankle problems;

5. finger, thumb, wrist, or hand problems; and,

6. elbow problems, often referred to as “tennis elbow” regardless ofwhether tennis was involved.

In addition to those types of problems, various types of skinulcerations (typified by bedsores, also called decubitis ulcers) occurin various types of patients, especially patients suffering fromdiabetes, obesity, or other types of circulatory, metabolic, or“ambulatory” (walking) problems. These types of ulcers, which typicallyare called skin ulcers to distinguish them from stomach ulcers (and toidentify them as a class of ulcers that are readily visible on skinsurfaces), frequently involve damage to underlying tissues as well,including muscles, tendons, and ligaments. They occur most frequentlyaround the feet and ankles (because of circulatory issues), or on bodysurfaces that tend to be pressed against bedding materials for hours ata time during sleep, especially among the elderly.

In addition, treatments using the types of cell preparations disclosedherein appear to be ideally suited for new mothers, immediately afterchildbirth. For example, they may be able to help accelerate the healingof, and improve the appearance and functionality of, any internal andexternal cuts or tearing that occurred during, for example, a Caesariandelivery, episiotomy, or delivery of an exceptionally large baby.

In addition to those types of uses, stromal precursor cells also can beuseful for reconstructing or altering the appearance of various types ofscars, and for similar types of surgery which can be generallyclassified as cosmetic surgery (i.e., surgery, injections, or otherinterventions in which alterations to appearance are of primary or majorimportance). A useful example can be offered by the repair of so-called“cleft palates”, in children who are born with a “missing tissue” typeof defect in their upper lips. Those and numerous other types of scars,defects, or irregularities can be rendered less noticeable and obvious,by injecting additional material into either: (i) a “deficit” type oflocation, in order to effective fill up that deficit; or, (ii) adjacentlocations, if the goal is to reduce the appearance and distractions ofan irregular and unwanted crest, promontory, or similar problem.Similarly, cosmetic alterations are often performed on people who havereached middle age or older, to reduce or reverse various types ofgradual facial or other tissue changes which accumulate as people agethrough adulthood.

Accordingly, the types of “stromal precursor cells” which have alreadyreached a state of partial differentiation, and which can complete amaturation process that will convert them into muscle, tendon, ligament,or other connective tissue cells, are of great interest, for the typesof medical treatments described herein. Stromal precursor cells fromautologous fatty tissue can be used to help repair muscles, tendons,ligaments, and other connective tissues that have been injured orotherwise damaged in various ways, or which have been surgically removedor otherwise manipulated (such as during removal of a tumor, cyst, orother injured, damaged, or unwanted tissue). They also can be used tohelp repair, regenerate, or replace tissue that was damaged byinfection, trauma, disease, or other events, conditions, or problems, orwhich cause unwanted displays of aging. Accordingly, if properlyperformed, these types of autologous transplantations of stromalprecursor cells, from fatty tissue, can help patients recover fromsports injuries, injuries suffered during various types of accidents ortrauma, infections by pathogenic microbes that attack connectivetissues, and similar problems. They also can be used for various typesof cosmetic and/or reconstructive surgery, such as for scar revision,repair of cleft palates, removal or reduction of skin lines and creases,etc.

Importantly, those types of stromal precursor cells are present insurprisingly high numbers, in fatty tissues, because of an importantcellular and physiological process.

Apoptosis; Active and Ongoing Cell Replacement in Soft Tissues

As mentioned above, because of a crucially important and highly activecellular process that occurs in all mammals, there are surprisinglylarge numbers of stromal precursor cells, in fatty tissue. While it isnot crucial to fully understand the natural cellular process in order tounderstand this invention, a working knowledge of it can help the readerreach a better understanding of how and why this invention works.

Cells that reach an advanced stage of aging are often referred to as“senescent”, which derives from the same root word as “senile”. In thesame way that a senile person might be able to live for many years, ifproperly taken care of by other people, senescent cells remain viable,as living cells, but they have lost the ability to fully perform theirnormal functions.

Severe health problems and major biological inefficiencies would arise,if muscles or other tissues in an animal had to devote part of theirresources to nurturing, nursing, and providing for senescent cells, inan effort to keep them alive for as long as possible even after theylost the ability to perform their essential functions. To avoid thoseproblems, mammalian cell biology evolved in a different direction.Rather than keeping cells alive for as long as possible, even after theycan no longer perform their essential functions, mammalian biologyevolved in ways that rapidly identify, kill, recycle, and replace agingand senescent cells with new cells, in a constant process of cellturnover and replacement. This process involves a well-known cellularactivity called “apoptosis”, sometimes referred to as “programmed celldeath”. This process is controlled by mitochondria, which are tiny“organelles” that have their own membranes, inside mammalian cells.These organelles are the remnants of tiny anaerobic bacteria, whichinitially invaded larger cells, and which then created a symbioticrelationship with their “host” cells. A typical cell has dozens or evenhundreds of mitochondria, inherited entirely from the mother, with nogenetic contribution from the father. These organelles are enclosedwithin their own membranes, and they carry their own DNA, which even hasits own specialized genetic code, which is different from the geneticcode used by the chromosomes in the nucleus of a cell.

Mitochondria are the “cellular furnaces”, where glucose (the main energysupply for any mammalian cell) is oxidized and “burned”, to convert theglucose into carbon dioxide and water, in a way that releases energywhich a cell can use. As a result, the chemical mixtures insidemitochondria are relatively harsh, since destructive “oxygen radicals”are constantly being generated, as a byproduct of glucose oxidation inthe mitochondria. As a result, the membranes which enclose themitochondria are chemically attacked and degraded, at relatively rapidrates.

In an aging cell, when the mitochondrial membranes become worn out anddegraded to a point where they become permeable, they begin releasing aspecific molecule called “cytochrome c”. That messenger moleculeactivates a sequence of events, which will culminate in a “macrophage”(a specialized white blood cell) engulfing, killing, and digesting theaging cell. This frees and releases molecular building blocks (such asamino acids, nucleotides, etc.), which will be used to make new cells,to replace the senescent cells that were killed and digested.

That type of “programmed cell death” is essential for keeping muscles,tendons, ligaments, internal organs, and other tissues vigorous andfully functional for spans of time measured in years or even decades.Except for neurons (which are in a special class), the typical lifespanof any particular cell, in any large animal, is only a few weeks ormonths, and the process which recycles and replaces aging cells with newcells, in any particular type of tissue, is very active at all times.

Therefore, the presence of large numbers of precursor cells which havereached a moderately advanced stage of development and differentiation,and which can complete the final steps of maturation into any particulartype of “adult” cell which is needed in some particular location at aspecific time, is crucially important to the process of constantlyreplacing aging cells with new cells. In any organ, joint, or other“subassembly” which contains multiple cell types, a rich supply ofpartially-differentiated precursor cells, which required a substantialamount of time to reach that stage of development but which can rapidlyundergo the last and final steps in a differentiation and maturationprocess that will create fully “adult” cells, is an essential part ofthe natural process of replacing old cells with new cells.

That is a well-known feature of mammalian physiology, and over the pastdecade, major advances have been made that allow physicians to extractand obtain large numbers of stromal precursor cells, from fatty tissuesthat can be obtained in a minimally-invasive manner, via liposuction.

Conventional Liposuction Procedures and Equipment

Conventional and well-known methods can be used to obtain stromalprecursor cells from fatty tissue, via liposuction. These methods aretaught in courses that are taken by surgeons and other doctors who wishto learn to perform these procedures, and the types of cannulae,syringes, and other kits, devices, and machines that are used duringthis type of liposuction are readily available. For example, a websiteat www.viafill.com illustrates the VIAFILL™ system, sold by the LiposeCorporation and specifically designed for the type of liposuctiondescribed herein.

Rather than resembling the types of enlarged cannulae that are used toremove large amounts of fatty tissue for the purpose of weightreduction, a cannula designed to harvest viable stromal cells forautologous transplantation has dimensions that are similar to anenlarged hypodermic needle. This type of cannula is made of a rigidmetal alloy, and has a smooth rounded tip that will not readilypuncture, cut, or damage muscles or membranes. A series of medium-sizedholes (or orifices, channels, or similar terms) pass through the sidesof the barrel, in a region near the tip of the cannula.

Before this type of cannula is inserted into a patient's body, ahypodermic needle (with a very thin barrel, and a very sharp beveledtip) is used to inject a topical anesthetic (such as xylocaine) underthe skin, at the location that will be worked on. Typically, the firstanesthetic needle is withdrawn, and after the first batch of anestheticdrug has taken effect and partially numbed the area, a second hypodermicneedle is inserted, and moved around a semi-circular area, to spreadadditional anesthesia into the area beneath the skin (this is oftencalled a “fanning” procedure or pattern, since the affected arearesembles the shape of a semi-circular hand-held fan). The needle iskept nearly tangential to the skin, so that the tip does not penetrateinto any major muscles, and remains in a shallow layer of fat betweenthe muscles and the skin.

Typically, as the second anesthetic needle is being withdrawn, the sharptip is used to create an enlarged nick in the skin. The smooth roundedtip of an injection cannula (which closely resembles or which can beidentical to the extraction cannula) is inserted through that nick inthe skin, into the fatty layer. That cannula is used to inject abuffered saline or similar aqueous solution into the fat, to helpliquefy the fat. This increases the quantity of fat (and the number ofviable stromal cells) that can be extracted from a relatively smallregion beneath the skin. The aqueous liquid emerges from holes in theside of the cannula, near the tip, and by using a combination of liquidinjection and cannular motion, the physician can create, in a relativelybrief time, a region of liquefied fatty tissue that is ready forextraction.

When that point is reached, the injection cannula is withdrawn, and anextraction cannula is inserted in its place. To minimize any risk ofunwanted complications or damage to tissue in the surrounding region,most surgeons operate an extraction cannula solely by using their handsto exert tension on the plunger handle, within an extraction syringe,without using a pump or other machine to create an artificial suction.By closely watching the flow of viscous fluid into the clear-walledbarrel of a syringe while sustaining a continuous and reliable “feel”(through their hands) for what the cannula is doing beneath the skin, askilled surgeon can develop a reliable sense of how to extract asubstantial volume of fluid in the safest possible manner, with minimalscarring, tissue disruption, or unwanted alterations or deformation ofthe skin surface contour in the affected region.

The only mechanical device which a surgeon typically uses, to helpsustain a continuous and relatively stable level of suction force on theextraction cannula, is a clip-type device commonly called a“JOHNNIE-LOK” (that phrase apparently is used as a trademark, by acompany called Tulip, but a search of the US trademark databaseindicates that any registration for that mark died in 2002). By means ofa simple twisting motion, this type of clip (which rests and pressesdirectly against the syringe opening) can be used to temporarily securethe plunger handle to the syringe barrel, at any position along thelength of the plunger handle. Accordingly, a surgeon can pull out theplunger handle until a desired level of suction force is reached, andthen use the “Johnnie-Lok” clip to sustain that level of suction, forsome period of time, while the surgeon focuses on the movement,positioning, and “feel” of the cannula tip beneath the skin. When enoughfluid has entered the syringe barrel to cause the suction force to dropto an undesirably low level, the surgeon twists the plunger handle torelease it from the “Johnnie-Lok”, pulls the plunger farther out of thesyringe barrel to re-establish a desired and effective level of suction,and then clips the plunger handle to the syringe at the new position.

This suction process continues until the syringe is nearly filled withliquid. At that point, the full (or loaded, etc.) syringe is removed,and a new and empty syringe is put in its place, to extract more fluid.This replacement can be done in either of two ways: (i) by detaching afull syringe from the extraction cannula while leaving the cannula inplace, in the patient's body; or, (ii) by pulling out the cannula, andreplacing it (this is often done is a surgeon suspects one or more ofthe holes in the cannula have become clogged).

As many syringes are used as are necessary to remove a quantity ofliquefied fatty tissue that the surgeon believes to be useful anddesired for a particular procedure on a particular patient. Thesevolumes are important, and they are discussed in more detail below,because they factor heavily into the specific teachings and claims ofthis invention.

Returning to background information which can help explain how thisinvention works, fatty tissue is a complex mixture which consists mainlyof four types of material:

(1) collagen, an extra-cellular fibrous protein which forms athree-dimensional “matrix” or “scaffold” which holds the cells togetherin essentially all connective tissues;

(2) large numbers of living cells, most of which will be attached or“anchored” to the extra-cellular collagen matrix;

(3) aqueous fluid, which comes from two main sources: (i) the salinesolution or other artificial fluid that was injected into the extractionsite, to help liquefy the fatty tissue; and, (ii) thenaturally-occurring aqueous fluids that are present even in fattytissue, mainly in the form of lymph and “tissue gel”, both of which helpnutrients reach and permeate into cells, and help carbon dioxide andother wastes diffuse away from the cells; and,

(4) a compound called “glycogen”, which is a polymerized fatty compoundused by mammals for energy and food storage. As described in anytextbook on physiology, glycogen is created by “stringing together”molecules of glucose (a specific 6-carbon sugar, which can be readilyand rapidly metabolized by all mammalian cells). When additional energysupplies are needed, the body can begin cleaving off individual glucosemolecules, one at a time, from strands of glycogen. Each molecule ofglucose can then be metabolized by the process called “glycolysis”, inwhich glucose molecules are “burned” as a fuel source, in a manner whichoxidizes the glucose into carbon dioxide and water, and which releasesenergy during the oxidation process.

Accordingly, when fatty tissue is converted into liquefied form (withthe aid of injected saline solution or similar liquid), for extractionby a cannula, collagen fibers must be broken, and a relatively thick andviscous mixture is removed, which contains still-living and viablecells.

There are two main types of uses for that type of fatty tissue, after ithas been extracted. One is for breast augmentation, in which carefulplacement of volume and bulk is the most important factor for achievinga desired cosmetic effect. The cell-concentrating procedures andimplantation methods described herein can be adapted for such use, ifdesired (especially for use in reconstructive surgery after a lumpectomyor breast removal, in a patient suffering from breast cancer, which doesindeed involve “connective tissue repair” as that term is used herein).Either of those types of surgery (i.e., breast enlargement for purelycosmetic purposes, or breast reconstruction following tumor removal) arespecialized types of surgery; they are well-known to cosmetic surgeons,and they can be carried out by surgeons who specialize in that type ofsurgery, using methods and cell preparations as disclosed herein, ifdesired. However, that type of cosmetic surgery is not of primaryinterest herein.

Although that type of cosmetic surgery can adapt and utilize theprocedures and equipment described herein, the primary and central focusof these types of treatments will involve connective tissue repairs, aslisted and summarized above. Accordingly, the discussion herein focuseson those types of uses.

Before the details of the invention herein are disclosed, one other typeof concentrated cell preparation, which was developed years ago andwhich is well-known and widely used, merits attention.

Combining Stromal Precursor Cells with “Platelet Rich Plasma”

It was discovered years ago (e.g., Abuzebi et al 2001) that the use ofstromal precursor cells, obtained from fatty tissues, will generatebetter results in connective tissue repairs, if the stromal cells aremixed or otherwise co-administered simultaneously with a blood-derivedpreparation called “platelet rich plasma” (PRP). Machinery and equipment(including “kits” with all necessary disposable devices and supplies)for creating PRP, from a patient's blood, are readily available tophysicians, as relatively small “desktop” devices that are roughly thesize of a typical microwave oven in a home. Accordingly, this aspect ofthe invention is old and known, and this section merely provides anoverview of platelet-rich plasma preparations from blood.

Platelets are specialized blood cells, which can be obtained fromcirculating blood. In mammals, they are heavily involved in the naturalhealing processes which arise in response to any type of injury.Accordingly, they were recognized by at least the 1980's as a rich andavailable source of: (i) growth factors, including hormones that willtrigger cell reproduction; (ii) proteins that can recognize and bind tothe “torn ends” of collagen fibers, at the site or a wound or otherinjury; and, (iii) other biomolecules which can help promote andaccelerate healing.

Briefly, when “whole blood” is processed (“fractionated”) in acentrifuge, at an appropriate speed which will not damage or kill bloodcells (often expressed as a multiple of “gravity” force, such as 40 g,which is commonly used in blood fractionation), three main layers willform.

The “bottom” layer will contain red blood cells (RBC's, also known aserythrocytes), since RBC's have the highest weight-per-volume density ofany of the blood components. Red blood cells do not contain anychromosomes; they are formed by a rapid process of cell “budding”,rather than cell replication, and they last for only a few days afterthey are created. Since their sole functions are to deliver oxygen andremove wastes, which require active circulation of blood, they wouldonly clutter and clog up an injured area which has been packed with astationary stromal-and-platelet cell mixture, for connective tissuerepair as described herein. Therefore, the red blood cells arediscarded, when a PRP mixture is being created for use in wound healing.

Above the bottom layer of red blood cells, a center layer will form,which mainly contains platelet cells, and white blood cells(leukocytes).

The uppermost layer, which is relatively clear but with a yellowishtint, contains the carrier liquid, which is blood plasma. If bloodplasma is further processed to remove fibrinogen and other clottingfactors (which are extra-cellular proteins), the resulting liquid iscalled blood serum.

In a broad sense, any blood preparation in which the plateletconcentration has been enriched above a “baseline” level (as found inunprocessed “whole blood”) can be referred to as “platelet rich plasma”(PRP). However, in order to render PRP truly effective for acceleratingand enhancing wound-healing activity, the concentration of plateletspreferably should be increased to at least about 5 times (5×) their“baseline” level.

After an initial centrifugation step has been used to remove red bloodcells from a processed blood fraction, other processing steps (includingvarious types of filtration, gel processing, “affinity binding”, orchromatographic steps, and possibly involving the addition of variousother agents) can be used, if desired. Depending on the specific stepsthat are used, this type of additional processing can, for example: (i)remove other types of white blood cells from a concentrated plateletpreparation; (ii) remove one or more specific targeted proteins (such asgrowth factors, albumin, etc.) or other molecules; (iii) remove aportion or fraction of the plasma liquid, from the platelets, to furtherincrease platelet density and concentration levels; or (iv) add varioussupplemental agents to a PRP preparation.

These types of processing steps, which can be used to create PRPmixtures for use in wound-healing procedures, are described in numerousreview articles, including Pietrzak et al 2005, Maniscalco et al 2008,Hall et al 2009, Gociman et al 2009, Lacci et al 2010, andLopez-Vidriero et al 2010.

Various companies sell processing machines that are specificallydesigned to extract PRP, from whole blood. A preferred system which hasbeen used with good results by the Applicant herein is the SmartPReP™Platelet Concentrate System, sold by Harvest Technologies. It isillustrated and described at www.harvesttech.com and in additionalsources which can be identified and obtained from that website. Thatsystem generates a PRP extract with high numbers of platelets andmonocytes, which are beneficial for the types of treatments describedherein, and with reduced numbers of other white blood cell types(including granulocytes, which are not especially beneficial forconnective tissue repairs as disclosed herein).

Other companies also make machines which can generate PRP extracts fromwhole blood; one example is the MAGELLAN™ system, sold by theArteriocyte Company (http://arteriocyte.com).

Both types of machines were introduced into the marketplace in the U.S.in 2009, and those two companies are competing against each other tocreate superior machines. Accordingly, various enhancements in suchmachines have further optimized PRP extraction methodology, and likelywill continue to do so.

In summary, methods which utilize mixtures and combinations of:

(1) stromal precursor cells, obtained from fatty tissue via liposuction;and,

(2) platelet cells, obtained and concentrated from blood samples, havebeen known and in use for more than a decade, for connective tissuerepairs. These methods have established an important specialty withinthe field of medicine and surgery, and the current invention involvesmethods and devices which can enhance and improve those surgical andmedical interventions and procedures.

Accordingly, one object of this invention is to disclose and providemethods and devices that can help achieve greater efficiency and betterresults, when stromal (connective tissue) precursor cells are obtainedfrom fatty tissue, via liposuction, for autologous transplantation intothe same patient, for connective tissue repairs.

Another object of this invention is to disclose and provide certaintypes of specialized devices, which include both reusable and disposablecomponents that will interact with each other, which will enableimproved methods of centrifugation and other processing steps, forhandling and concentrating stromal precursor cells which will be usedfor autologous transplantation.

Another object of this invention is to disclose methods and devices forcarrying out a process of concentrating and enriching autologous cellsthat have been harvested from fatty tissue extracted from a patient vialiposuction, which will minimize any loss of viable stromal precursorcells from the fatty tissue extract.

Another object of this invention is to disclose methods and devices forconcentrating stromal precursor cells that have been harvested from apatient, which involve mechanical rather than enzymatic or otherbiochemical processing, and: (i) which can be completed quickly, such aswithin 20 minutes or less, rather than requiring a sustained incubationor reaction period to complete an enzymatic treatment; and, (ii) whichminimize or eliminate the types of governmental scrutiny and approvalrequirements that come into play when chemical treatments are performedon cells which will then be permanently implanted in a patient's body.

These and other objects of the invention will become more apparent fromthe following summary, drawings, and detailed description.

SUMMARY OF THE INVENTION

Methods and devices are disclosed for processing stromal precursor cells(i.e., cells which can differentiate into various types of connectivetissue cells, such as cells in muscles, ligaments, tendons, etc.), whichcan be obtained in large numbers from fatty tissue extracts, obtainedvia liposuction. These types of cells, either by themselves or whencombined with a second cellular mixture called “platelet-rich plasma”(PRP), can be used for autologous cell transplantations, to repair,regenerate, or supplement connective tissue, at an injury site or otherlocation that is in need of repair or other medical intervention.

Processing of a fatty tissue extract involves centrifugation during aninitial separation step, to concentrate stromal precursor cells fromliposuction fluid into a semi-concentrated form that is usually called“spun fat” in the prior art.

The “spun fat” should then be treated by a second stage of processing,as disclosed herein, to further concentrate the stromal precursor cellswhile eliminating glycogen, fat, and remnants of the extra-cellularcollagenous matrix. In one embodiment, the cell suspension can beincubated with collagenase, an enzyme, to break down and remove theextra-cellular collagen fibers that will be present in the initial fattyextract. However, an alternate embodiment is strongly preferred overcollagenase treatment, for reasons described below. In the preferredprocess, mechanical means (such as mildly forcing the “spun fat” cellsuspension through an extrusion device) can be used to detach stromalprecursor cells from the extra-cellular collagen matrix. A secondcentrifugation and/or filtration step can then be used to furtherconcentrate the stromal precursor cells that have been released from thefatty matrix of the spun fat material.

By means of these processing steps, stromal precursor cells from a fattyliposuction extract can be converted into a highly concentrated form,for subsequent reintroduction (along with platelet-rich plasma, ifdesired) into a wound site or similar area that is in need of tissuerepair.

In addition to disclosing those type of enzymatic and/or mechanicalmethods for extracting concentrated stromal cell preparations fromliposuction extracts, specialized centrifugation cartridges aredisclosed herein, which will enable up to 120 cubic centimeters (cc) offluidized liposuction extract, in six syringes which will hold 20 cceach, to be processed in a single centrifugation step, using the samecentrifuge machine that is also used to prepare platelet-rich plasma(PRP). To accomplish that, each 20 cc syringe must be short enough tofit into an accommodating centrifugation cartridge which will containthree wells, to hold up to three syringes at a time. These types ofcentrifuges hold two cartridges at a time, at opposite ends of a rotor,for proper balance during high speed centrifugation. Accordingly, a setof two cartridges, holding three syringes each, will hold six syringes,containing up to 120 cc of liposuction extract with stromal precursorcells. It has been found that 120 cc of fatty tissue extract issufficient to satisfactorily handle the very large majority of suchprocedures, even in cases where a patient will require a series ofmultiple cell injection procedures over a span of multiple months.

Furthermore, the new types of centrifugation cartridges disclosed herealso can be provided with a lipophilic absorbent material insert, whichwill withdraw and sequester the liquefied oils which are contained in a“spun fat” preparation. It has been found by the Applicant herein thatthose liquefied oils become toxic to stromal precursor cells, when theoils become concentrated to the point that occurs in spun fat.Therefore, a component or process which removes those oils fromsustained contact with the cells will help increase the health andviability of the cells that can be returned to a patient's body.

Accordingly, improved methods and devices are disclosed herein, forrapid and convenient preparation of highly purified stromal precursorcells, for use in connective tissue repairs. In addition, an improvedcombination of centrifuge cartridges and liposuction syringes aredisclosed herein, to provide a single set of devices that will enableconvenient and efficient handling of up to 120 cc of liposuction extractat a time, using the same centrifuge machine which is used to prepareplatelet-rich plasma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (which is prior art) depicts of the layers that will be formed,when a liquefied fatty tissue extract, obtained from a patient vialiposuction, is centrifuged for a suitable period of time at a speedthat will not damage viable cells. This drawing shows two identicalsyringes, each containing approximately 20 milliliters (mL or ml) of afluidized liposuction extract which has been centrifuged; both syringesare held within accommodating wells, in a cartridge designed for use ina centrifuge machine. The uppermost oily layer (with the lowest density)will be discarded. The center layer, referred to herein as “spun fat”for convenience (and as “concentrated fatty tissue extract” in theclaims) will contain a semi-concentrated suspension of stromal precursorcells (which can also be called connective tissue precursor orprogenitor cells). The bottom layer, which has the greatestweight-per-volume density, will contain mostly water, but it will alsocontain substantial numbers of stromal precursor cells; therefore, thatwatery layer will be forced, using gentle pressure and/or suction,through a filter which does not allow cells to pass through. This willretain the stromal precursor cells on the surface of the filter; theycan then be washed off of the filter surface, and mixed with cells inthe “spun fat” layer, for additional processing.

For comparative and descriptive purposes, the syringes on the left sideof FIG. 1 is shown in a well that has a valve-controlled flow conduitwhich leads to a filtering device. The syringe on the right side isshown with a simple “cap” screwed to its tip. The first arrangement isfeasible; however, the second arrangement is generally preferred, sinceit allows the syringe to be simply removed from the centrifuge deviceand then handled conveniently.

FIG. 2 is a flow chart depicting a series of steps that can be used toprocess, extract, and utilize stromal precursor cells contained in aliposuction extract from a patient who requires connective tissuerepair.

FIG. 3 is a schematic depiction of a device for mechanically separatingstromal precursor cells from fat and collagen fibers, in a “spun fat”suspension created by: (i) initial centrifugation of a liposuctionextract, followed by (ii) mixing the spun fat material with a salinebuffer containing a gentle surfactant, such as lecithin. A piston orplunger is used to force the cell suspension downward, through anextrusion plate having multiple small tapered orifices passing throughit. This will partially break apart the spun fat layer, in a manner thatwill begin releasing the stromal precursor cells from the fat andcollagen matrix. The mixture of water, fat, and cells is circulated androtated, with the aid of a mechanical stirring device, around acylindrical chamber having several perforated “catch plates” (forsimplicity, only one such catch plate is shown in FIG. 3). Fatty andoily droplets in the aqueous suspension will impinge against and clingto the catch plates, while the stromal precursor cells will drop out ofsolution and accumulate on a sloped floor of the cylinder, for removalvia an outlet.

FIG. 4 is a schematic depiction of a “screen passaging” system which canuse vibrating, reciprocating, or tapped screens, to mechanicallyseparate viable cells from the fat and collagen matrix in a “spun fat”suspension created by centrifuging a liposuction extract.

FIG. 5 is a schematic depiction of a “shearing-force stirring” system tomechanically separate viable cells from the fat and collagen matrix in a“spun fat” suspension created by centrifuging a liposuction extract.

FIG. 6 is a schematic depiction of an “extruding and centrifuging”device which can fit inside a centrifuge machine, and which uses adisplacing device comparable to an inflatable balloon catheter, togently force the “spun fat” preparation through small holes which passthrough a cylindrical wall, to reach an annular collection space,thereby gently prying stromal precursor cells away from collagen fibersand extra-cellular debris, so that the cells can be separated by acentrifugation step using the same extruding device.

FIG. 7 is a schematic depiction of the same device shown in FIG. 6, alsoshowing an oil removal component which can extract and sequesterliquefied oils that will emerge from the “spun fat” material, so thatthose oils will not remain in sustained contact with the stromalprecursor cells, which would damage the cells by clogging and foulingtheir surface receptors.

FIG. 8 is a schematic depiction of an “extruding and centrifuging”device which uses a large rotatable threaded shaft to displace and forcespun fat through an extrusion tube.

FIG. 9 is a schematic depiction of an “extruding and centrifuging”device which uses centrifugal force to drive spun fat through acone-shaped extruder.

FIG. 10 is a schematic depiction of an “extruding and centrifuging”device which uses centrifugal force to drive spun fat through an entireset of annular and planar extrusion surfaces.

FIG. 11 is a schematic depiction of an “extruding and centrifuging”device which uses centrifugal force to drive spun fat through adual-mesh system, to separate stromal precursor cells fromextra-cellular material.

FIG. 12 is a schematic depiction of an “extruding and centrifuging”device which uses centrifugal force to drive spun fat through a layer oflightweight beads, so that the jostling activity, as heavier cells swappositions with lighter beads, can help separate stromal precursor cellsfrom extra-cellular material.

FIG. 13 is a perspective view which depicts a centrifugation cartridgethat can hold three syringes, with each syringe holding up to 20 cc involume. This centrifugation cartridge is sized and designed to fit intoa commercially available centrifuge machine that is designed and suitedfor preparing platelet-rich plasma (PRP) from whole blood.

FIG. 14 is an elevation (plan) view of two centrifugation cartridges,each having three wells for syringes that can contain 20 cc each ofliposuction fluid. Each cartridge is held within a basket device at oneend of a rotor, in a centrifuge. The wells in the cartridge are labeled,to indicate a loading sequence that will maintain balance and symmetryregardless of whether two, four, or six syringes are loaded into thecentrifuge. A small additional well is also included in each cartridge,which can hold water or any other liquid or weight, to evenly balancethe weights of two cartridges (including any loaded syringes held by thecartridges) against each other.

DETAILED DESCRIPTION

As summarized above, this invention relates to improved devices forprocessing and concentrating stromal precursor cells (i.e., cells whichcan mature into connective tissues, such as muscles, tendons, andligaments). Those types of cells can be obtained from fatty tissues in apatient's body, by means of liposuction. If processed properly, thosetypes of precursor cells can be extremely useful and helpful intreatments for various types of tissue defects, including but notlimited to treatments for injuries, infections, arthritic or similardegeneration, for scar revision, or for other problems that areassociated with joints, limbs, or other connective tissues.

If desired, stromal precursor cell preparations as described herein canbe mixed with concentrated preparations of specialized blood cellscalled “platelets”, such as in a liquefied preparation called“platelet-rich plasma” (PRP), which is well-known in this field of art.When a concentrated preparation of stromal precursor cells is mixed withplatelets (which specialize in promoting tissue repair, and the mixtureis injected into an injury or infection site, the result can becomparable to having both: (i) a set of skilled carpenters (which areanalogous to the platelet cells), and (ii) a supply of buildingmaterials (which are analogous to the stromal precursor cells), at a jobsite where something needs to be built.

Conventional liposuction procedures, which have been used with goodresults by the Applicant herein to obtain supplies of stromal precursorcells from patients, are described in Example 1. However, this inventiondoes not depend upon, arise from, or claim any specific procedures ordetails relating to any process or method used to extract fatty tissuefrom a patient. When human patients are involved, liposuction (or anyother form of adipose tissue extraction) is a form of surgery, and canbe performed only by qualified and licensed physicians. Any physicianwho performs liposuction will have his or her own preferences concerningequipment, methods, and kits with disposable supplies, for performingliposuction.

Various devices and supplies for performing low-volume liposuction(i.e., the type of liposuction procedures that are suited for harvestingstromal precursor cells, as distinct from high-volume liposuction, asused for weight reduction purposes) have been available for years, andare well known to those skilled in this field of surgery. As oneexample, the VIAFILL™ system, sold by the Lipose Corporation, includesvarious tools and instruments (generally referred to as “devices”herein, to distinguish them from liquid-type supplies, reagents,pharmaceuticals, etc.) that are specifically designed for low-volumeliposuction. Those are described and illustrated at www.viafill.com.

However, there is one factor that requires attention, with regard to theliposuction part of the treatments described herein. This factor relatesto the volumes of fatty tissue that should be extracted, for carryingout various different types of treatments.

Liposuction Extraction Volumes

Based on trials and tests on a number of patients (all of whom providedinformed consent), which involved treatments for some of the most commontypes of connective tissue injuries that can benefit from stromalprecursor cell transplantations as described herein, the Applicantherein has determined that volumes such as those listed in Table 1usually are appropriate in most cases of typical pain, discomfort, anddamage, when treating an adult male. These volumes will include aqueousfluids that were injected into a patient to help break apart and liquefythe fatty tissue that was being removed, and which was then suctionedout along with the fatty tissue. Accordingly, volumes of actual fattytissue which are extracted during these procedures, when injectedaqueous fluids are not included, are correspondingly lower. Furthermore,when treating cases of severe damage and pain, correspondingly largervolumes should generally be extracted, in anticipation that a series ofseveral injections may be required, over a span of months, to providebetter results.

TABLE 1 TYPICAL FLUID EXTRACTION AND PROCESSED CELL VOLUMES LiposuctionExtract Processed Stromal Cells Type of Treatment (cc) (cc) Shoulder(rotator cuff) 30-50 3 Elbow (tendonitis) 30-50 3 Hip 50-80 4-6 Knee50-80 4-6 Ankle 30-50 3

Table 1 also indicates the approximate volumes of fully-processedstromal precursor cells which should be obtained via these types ofliposuction procedures, when the cells have been fully processed andconcentrated, prior to injection back into a patient. These concentratedcell volumes will be contained in a cellular pellet or layer, which willbe generated as described below and as indicated in FIG. 2.

If it appears that inadequate numbers of cells are present in the fattytissue extract from some particular patient, then the treating physiciancan extract additional fatty tissue, either from the same area that hasalready been anesthetized, or from a different area of the patient'sbody (such as (i) if the anesthesia has partially worn off, and/or (ii)if the withdrawal of additional fatty tissue from the initialliposuction site might leave undesirable surface irregularities). If aphysician wishes to do so, he or she can check the apparent cell densityper volume of fluid, in one or more small samples of fluid taken fromthe “spun fat” material after the first centrifugation, or from a sampleof liquid taken at any stage during subsequent processing. That type ofcheck, for cells-per-volume density, can be performed using a lightmicroscope if desired, or by means of more sophisticated equipment, suchas a flow cytometer.

An important factor which must be taken into account, when planning andperforming liposuction to obtain a sufficient quantity of stromalprecursor cells for any particular treatment, arises from the fact thatcertain types of injuries can require two or more treatments, atdifferent times, to obtain optimal results. Such repeated treatmentsnormally will involve: (i) a first injection of stromal precursor cells(which can be mixed with PRP if desired), usually on the same day thatthe liposuction is performed; and, (ii) one or more additionaltreatments, which typically will occur at least a month or more afterthe initial treatment.

For example, if a patient requires “bilateral” treatments (i.e., on bothsides of the body, such as on both hips, or both knees), a preferredapproach usually will involve: (1) treating a first hip or knee, whichusually will involve the joint that is causing the most pain ordiscomfort at that time; and (2) giving the patient 2 or 3 months forthat treatment to take full effect, while the patient undergoes atraining, rehab, strengthening, or similar program which will focus onthe treated joint, without any interference or impairment that wouldoccur if both joints on both sides were treated in a single session.Subsequently, after the initial treatment on one side has fully settledin and stabilized, the second joint will be treated, and that secondtreatment will require its own rehab, strengthening, or similar programfollowing the treatment.

Other situations also arise in which stromal cell treatments willprovide greater benefits if repeated one or more times. For example, anumber of patients who suffered from various types of problems (includethe types of arthritis that involve damage to cartilage, in joints suchas knees or hips) benefitted from a series of two or more treatments asdisclosed herein, at the same treatment site. Patients in this categorycommonly reported that an initial treatment provided a large measure ofrelief, such as by eliminating 60 to 90 percent of the initial pain.Those patients chose to undergo a second treatment, to determine whetherthe second treatment could provide even more relief. Accordingly,follow-up treatments for patients in this class can be regarded ascomparable to “booster” shots. When these types of repeated treatmentsare involved, the entire series of treatments, taken together, willrequire a larger volume of liposuction extract.

It also should be noted that pellets or layers of processed andcompacted stromal cells, prepared as described herein, have been storedfor up to six months in medical-grade freezers (which can sustaintemperatures such as −80° C., which is in the temperature range ofliquid nitrogen), with no signs of cell damage or loss of viability.

Accordingly, based on those initial tests that have been performed todate, the Applicant herein has discovered that if liposuction is used toextract liquefied fatty tissue in volumes of up to 120 cc (which willinclude a significant quantity of injected saline solution, mixed withthe fatty tissue), then that 120 cc volume will enable the treatingphysician to meet the needs of the very large majority (such as morethan about 95%) of all patients who are in need of these types oftreatments, including patients who need a series of repeated treatments.

Therefore, this invention discloses a set of centrifuge cartridges whichcan hold and accommodate six syringes at a time, with each syringeholding 20 cc of fluid, and with all of the syringes sized to fit into acentrifuge cartridge that will fit into a “desktop centrifuge” machine.

As indicated by the name, a “desktop centrifuge” has dimensions thatenable it to sit, in a stable manner at all times during loading,operation, and unloading, on top of a desk, table, laboratory-typebench, or similar furniture-type surface with a conventional height(such as from roughly mid-thigh, to slightly above waist-high, on anaverage adult), while providing full visual and working access to a lidor other movable top compartment which is low enough to allow an averageadult of normal height to lean over, look into, and load and unload themachine while standing normally on the floor next to the desk, bench,etc. By contrast, most “free-standing” centrifuge machines designed forfull-time laboratories are comparable in size to a washing machine.Numerous types of “desktop” centrifuges have been available for decades,and they are in widespread use in the offices and clinics of largenumbers of physicians.

There is no need for a centrifuge cartridge to be able to fit into twoor more different types of centrifuge machines, in order to meet therequirements of this invention. Instead, if a centrifuge cartridges withat least two or more wells that are sized and designed to hold 20 ccsyringes is sized and suited to fit in a stable and secure manner intoat least one type of conventional desktop centrifuge, then thatcentrifuge cartridge is regarded as being within the scope of theinvention and claims disclosed herein, provided that it meets all otherrelevant limitations of the claims. Accordingly, one or moremanufacturers can sell such cartridges as accessories, which can bepurchased by the user of any particular make and model of a desktopcentrifuge, who will know exactly which type of desktop centrifuge ispresent and available for use in any particular physician's office orclinic. This would be comparable to a car owner being able to purchasetires for any particular make and model of automobile, merely by tellingsomeone who works at the tire store the make and model of the car.

Alternately, rather than having to address potential issues concerningthe various different models of desktop centrifuges that are commonlyowned and used by physicians' offices and clinics, a preferred approachto designing and making centrifuge cartridges that will hold at leasttwo and preferably three 20 cc syringes can focus, instead, on certaintypes of specialized centrifuge machines that are specifically designedfor preparing “platelet rich plasma” (PRP). That class of specializedequipment underwent a major restructuring in 2009, when two specifictypes of desktop-sized PRP units, designed for physicians' offices andclinics, were commercialized: the SMARTPReP™ system, sold by HarvestTechnologies, and the MAGELLAN™ system, sold by the Arteriocyte Company.

Since the preparation of high-quality PRP (with platelet cellconcentrations at least 5× greater than baseline, and preferably withreduced granulocyte cell concentrations as well) is essential for thetreatments herein, and since both the SMARTPReP and MAGELLAN systemsinclude relatively compact and reasonably-priced desktop centrifugemachines as major elements of those systems, a useful workingpresumption is that the large majority of any physicians' offices andclinics which specialize in “sports medicine” and/or orthopedics willhave one of those two types of machines. Therefore, if syringe-holdingcentrifuge cartridges are manufactured and sold, which are designed andsized to hold at least two 20 cc syringes while fitting into one ofthose two types of PRP centrifuges, then having those simple optionswill enable any physician's office or clinic that has a platelet-richplasma (PRP) centrifuge, will be able to centrifuge up to 80 or even 120cc of liposuction extract at a time, using the methods disclosed herein,in the same desktop centrifuge that is used to prepare PRP.

A separate section, below, addresses the dimensions of centrifugecartridges for PRP machines, and for 20 cc syringes which will fit intothose types of cartridges. For now, the sections directly belowsummarize the major steps of the procedure, with reference to thedrawings.

Finally, it should be noted that the ability to centrifuge liposuctionextract fluid, while the fluid is still inside in the syringe that wasused to extract it rather than transferring it to a different vessel orcontainer for centrifugation, is highly useful and convenient, since itcan avoid the requirement for additional steps and safeguards that wouldneed to be developed and utilized, to absolutely minimize any risk ofcontamination and infection. Anything which can be done to minimize thenumber of items or surfaces which will contact any cellular or fluidmaterial that will be injected into a patient, or which can be done toensure the sterility of any such vessel, tubing, filter, and otherdevice which contacts any cells or fluids that are injected into apatient, can facilitate and improve the types of connective tissuerepairs described herein.

First Centrifugation Step: The Liposuction Fluid

FIG. 1 is a schematic depiction of two syringe barrels 80 and 90, heldparallel to each other in a syringe-holding centrifuge cartridge 100.For purposes of description at this stage, both of the two syringesbarrels 80 and 90 (which also can be called syringe cylinders, tubes, orsimilar terms, and which also are referred to herein simply as syringes,for convenience) are standard and conventional disposable plastic“monoject” syringes, as well known in the prior art. However, certaintypes of modified syringes, having wider diameters and shorter lengthsto enable three fully-loaded 20 cc syringes to fit into cartridges thatwill fit into specialized centrifuges designed for preparingplatelet-rich plasma, are also of interest herein, and are describedbelow.

In FIG. 1, each syringe 80 or 90 holds a suitable volume (such as 20 cc)of a liposuction extract 150, taken from a patient in a singleliposuction procedure, using methods such as described in Example 1. Theliposuction extracts depicted in FIG. 1 have been centrifuged at about40 G (i.e., at a rotational speed which will establish centrifugalforces that are 40 times greater than the force of gravity) for about 8to 10 minutes. It has been established and reported, by variousresearchers, that centrifugal forces of up to about 40 G, imposed for 10minutes at a time, will not substantially damage or kill the types ofstromal precursor cells that are involved.

As depicted in that drawing, the “bottom” (highest density) aqueouslayer 152, in each syringe, will contain a watery liquid. This layerwill be adjacent to a syringe tip 82 or 92. During high-speed rotation,the syringe tips 82 and 92 will travel around the outer periphery orcircumference of the syringe pathway, near the circular interior wall ofthe centrifuge chamber. The opposed ends 84 or 94 of the syringes areusually referred to as the opening, throat, or similar terms.

The temporary coupling of a syringe tip to a liposuction extractioncannula, during liposuction, can use a conventional “luer” fitting. Thatname, which came from a 19th century German instrument maker who playeda major role in the early development of these devices, refers tostandardized fitting sizes that are used for small fluid-handlingdevices. Luer fittings are divided into two major classes. So-called“luerlock” fittings (also spelled luerlok) use accommodating threads,which are enlarged (compared to typical screw threads), to make themeasy to engage, and so that only about 2 rotations are required to fullyand securely couple a syringe tip to a cannula or other device.

Other types of “luer” connectors which are not threaded are often called“luerslip” systems. These usually are adequate for small couplings thatare used only briefly and that do not need to withstand substantialpressures. However, since any liposuction syringe as discussed hereinwill need to withstand centrifugation for a prolonged time, luerslipconnectors would not be well-suited for such syringes. Accordingly,syringe tip 92 is shown as having luerlock threads, which have beenscrewed tightly into a small internally-threaded cap 99, forcentrifugation. Cap 99 will press securely into a conical or similar“floor” 102, which is provided as part of a well 104 which is designedto hold syringe barrel 90.

Returning to the liquefied material contained in both syringes, the next“higher” layer 154, above aqueous layer 152, contains a thick, viscous,and relatively opaque fluidized or paste-like material, referred toherein as “spun fat” for convenience in this specification, and as“concentrated fatty tissue extract” in the claims. This “spun fat” layerwill contain large numbers of cells, along with glycogen, brokencollagen fibers, etc.

The uppermost (lowest density) layer 156 will be a generally clear layerof oily liquid, which will contain few if any viable cells. Oily layer156 will be discarded.

A movable mechanical plunger tip 86 or 96, made of rubber or a flexiblepolymer, will rest on top of the oily layer 122, near the opening ofeach syringe. It will form a movable but generally watertight sealbetween the outer rim of the plunger tip 86 or 96, and the smooth innersurface of the syringe barrel. During the liposuction procedure, aplunger or handle device (not shown), which is small enough to travelwithin the syringe barrel, will have its lower tip securely attached toa rubber plunger tip 86 or 98, by means such as a threaded fitting; thiswill allow the plunger handle to be secured to, or removed from, theplunger tip whenever needed.

During liposuction, the surgeon will exert a gentle but firm pullingforce (or a tensile or withdrawing force, or similar terms) on theplunger handle, to create a controlled level of suction inside thesyringe, to extract liquefied fatty tissue from the patient's body. Whena syringe barrel is sufficiently full of liquefied tissue, thefully-loaded syringe will be unscrewed from the cannula and replaced byan empty syringe, so that more liquefied tissue can be extracted. Thesurgeon can use as many syringes as desired, for any treatment on anyspecific patient. If desired, the cannula tip can remain inside thepatient, each time a full syringe is replaced by an empty syringe.However, if the surgeon suspects that one or more of the inlet holes ofthe cannula might be clogged, the surgeon can temporarily withdraw thecannula from the patient (the affected skin area will remainanesthetized and numb), to inspect the cannula and clean it ifnecessary.

After a syringe loaded with fluidized liposuction extract has beenproperly centrifuged (such as at about 40 G for 8 to 10 minutes), waterylayer 152 will be an aqueous suspension which will contain substantialnumbers of stromal precursor cells. The watery liquid itself wouldinterfere with wound healing, and should not be injected into an injurysite or other targeted treatment site; therefore, the water will beremoved from the stromal cell preparation, and discarded. However,rather than losing and wasting the stromal precursor cells that arecontained in aqueous layer 152, the watery layer 152 preferably shouldbe passed through a filter device, which will cause the cells to remainon the filter surface, while water passes through the filter, fordisposal. Various types of commercially-available cell filters areavailable, which will allow a watery liquid to pass through the filterwhile retaining any cells suspended in the liquid. Membrane filters madeof polysulfones, with pore diameters that average 13 to 15 microns, havebeen used by the Applicant herein with good results. Filter segmentswith suitable sizes (such as discs having 15 mm diameters) can be heldin any suitable holding mechanism.

To illustrate the type of cell-filtering devices of interest herein, afilter segment 170 is shown in FIG. 1, embedded within centrifugecartridge 100, and positioned for use with syringe 80. If filter 170 isembedded within cartridge 100, then at least two additional elementswill also be required:

(1) a conduit which will carry the aqueous liquid from the syringe tothe filter, after centrifugation;

(2) a valve device 172, which should be positioned between the syringetip 82 and the filter material 170, to prevent oily material fromcontacting and coating the filter surface during the centrifugation;and,

(2) a valve access port 174 (or similar means), to allow a physician orassistant to open and close valve 172 at appropriate times.

It also should be noted that if a filter and the necessary conduits andvalve are provided within a centrifuge cartridge, they will reduce the“effective length” of the cartridge, which will need to hold up to three20 cc syringes.

In addition, the risk that some quantity of oily and/or fatty material,from a liposuction extract, might contact and coat the filter media,before the cell filtering step has been completed, is anotherpotentially important problem. This risk is increased by the fact thatif a small “plug” of fatty material is positioned in the narrow tip 82of syringe 80, it can be difficult to dislodge and displace that “plug”of fatty material, with a slightly denser watery material, no matter howlong the liposuction extract is centrifuged, because of the way thefatty “plug” will be effectively forced and “wedged” into a small andnarrow channel which will appear to be a “dead end” duringcentrifugation. Contacting and possibly coating a porous filter witheven a small quantity of a thick and sticky oily material, beforepassing an aqueous solution through the filter, is not desirable, and insome cases it may pose a risk of seriously impairing or disrupting thecell filtering operation.

For those reasons, the filtering arrangement shown on the left side ofFIG. 1, while feasible, is not a preferred design, and it is presumedthat any cell filtration steps should, instead, be carried out using aseparate filtration device which can be optimized for such use ratherthan being “squeezed” into the tight confines of a cartridge that isdesigned mainly to hold at least two and preferably three large syringeswhile spinning in a centrifuge.

Accordingly, syringe 90 (depicted on the right side of FIG. 1) shows athreaded cap 99, which has been screwed onto the threaded tip 92 ofsyringe 90 during the centrifugation step. During centrifugation, cap 99will be pressed against a conical sloping (or other suitably shaped)“floor” 102, which will be provided as part of a syringe-holding “well”104 which is provided in cartridge 100. After centrifugation has beencompleted, syringe 90 (and cap 99) will be lifted out of the well, aplunger handle will be screwed onto rubber plunger tip 96, and cap 99will be unscrewed from syringe tip 92. Using the plunger, the aqueouslayer 152 will then be pushed out of the syringe barrel 90, and into afiltration device and through a filter material that will allow water topass through while retaining cells on the surface of the filter.

In one preferred embodiment, the filtration device can be a relativelysmall and lightweight device which can be turned either “right-side up”or “upside down”, at different stages of the work, to take advantage ofgravity-assisted flow during each of two different stages. When a wateryliquid containing stromal precursor cells is being passed through thefilter, downward flow of the watery liquid, through the filter, isbeneficial. However, after that step has been completed, when the timearrives to remove and recover the cells from the surface of the filterso that the cells can be processed for reinjection back into a patient,it becomes beneficial to use a “pulse” of water which travels in adownward direction through the filter. This will allow the washingliquid to dislodge cells from a bottom surface (or underside, or similarterms) of the filter, in a way that will allow the cells to simply falloff the underside of the filter, for collection in a small cup, basin,tray, or similar receptacle that is positioned beneath the filter duringthe filter-washing and cell-recovery step.

As mentioned below, if a collagenase digestion step will be used, anaqueous saline solution (such as “Hanks balanced salt solution”,abbreviated as HBSS) will be added to the “spun fat” layer, while thespun fat is being incubated with collagenase. Accordingly, that sametype of saline solution can be used to wash the cells off a filtersurface, when it is time to recover the filtered cells. The salinesolution that is used to wash cells off of a filter surface can be usedas part of the saline solution that will be added to the spun fat, tohelp promote a collagenase digestion step.

Alternately, if mechanical processing (rather than enzymatic digestion)will be used to dislodge and detach stromal precursor cells fromextra-cellular collagen fibers, then it should be kept in mind that anycells which became suspended in an aqueous layer 152, created bycentrifuging a liposuction extract fluid, very likely have alreadybecome detached from any extracellular collagen fibers, and thereforewill not need to be passed through an additional mechanical processingstep, which by its nature will necessarily inflict some level ofshearing and other stresses on cells being treated. Accordingly, if acell-containing filter-rinsing liquid is created, it likely should beheld aside and not included, when the mechanical processing step is usedto dislodge and detach stromal precursor cells from extra-cellularcollagen fibers. The aqueous-suspended filtered cells can be added backto the other cells from the spun fat layer, once the cells from the spunfat layer have passed through the mechanical processing and are readyfor final centrifugation.

After the initial centrifugation step involving the “raw” liposuctionextract has been completed, to form the layers depicted by FIG. 1, thesyringes (with threaded caps still attached to the threaded tips of thesyringes) will be lifted out of the centrifugation cartridge, and placedin a stationary holding device, to help separate and process the layersthat were formed during centrifugation. Any of several differentsequences of steps can be used for the separation steps, such as thefollowing:

(1) the plunger handle is reattached to the rubber plunger tip, whichremained at all times inside the syringe barrel;

(2) the threaded cap is unscrewed from the tip of the syringe;

(3) the syringe tip is coupled to a clear conduit or device (presumablya relatively thin flexible plastic tube) that will carry the water layerto a filter device, which will allow water to pass through the filterwhile retaining the stromal precursor cells on the filter surface. Ifdesired, it may be preferable to divert the first drop of fluid thatemerges from each syringe, onto an absorbent material that will bediscarded (such as a paper towel), so that if a small quantity of stickyfatty material formed a “plug” in the syringe tip during centrifugation,it will be diverted and disposed of, before it can contact and possiblycoat and foul the filter material;

(4) the plunger handle is pushed into the syringe, until the aqueouslayer has been expelled from the syringe barrel and passed through acell filter;

(5) the syringe is then uncoupled from the cell-filtering device whichhandles the aqueous layer, and is coupled to a different conduit ordevice;

(6) the plunger handle is pushed deeper into the syringe, to expel the“spun fat” layer from the syringe (it will be a viscous paste-likecompound, comparable to toothpaste) and force it into a chamber orvessel that is suited for: (i) a collagenase incubation step; (ii) amechanical processing step as described below; or, (iii) mixing with asuitable aqueous solution, such as “Hanks balanced salt solution” and/orlecithin or a similar surfactant, before a mechanical processing step asdescribed below;

(7) when the entire “spun fat” layer has been expelled from the syringe,the plunger handle can be pulled out of the syringe (with the rubber tipstill attached to it), and the low-density oily layer can be drainedand/or rinsed out of the syringe and discarded; alternately, the plungerhandle can be unscrewed from the rubber tip, and the syringe barrel,with the oily layer and rubber tip still inside, can be discarded asmedical waste.

If desired, the spun fat layer 112 (with a relatively small quantity offiltered cells from the aqueous solution) can be reinjected directlyback into the patient, along with platelet-rich plasma. This option isindicated by the lowest box on the left-hand side of FIG. 2, which is aflowchart that summarizes the cell processing steps discussed herein.That treatment method was used by the Applicant herein in a number ofearly trials, with generally good results when compared to conventionaltreatments known in the prior art.

However, as indicated by the alternate pathway in lower right quadrantof FIG. 2, a more extensive and thorough processing method can enrichand concentrate the stromal precursor cells to a substantially higherand better level, before they are injected back into the patient. Thoseadditional steps are described below. Because of: (i) the nature and thereadily-predictable results of this type of additional processing; (ii)the visible results on processed cells, when before-and-after resultsare analyzed under a microscope; and, (ii) results that the Applicanthas seen to date, in patients who have been tested in small-scale trialsof these methods, the Applicant herein regards these additionalprocessing steps as highly beneficial, to a point of clearly justifyingthe extra time and effort required by these additional steps.

Collagenase Incubation, Followed by a Second Centrifugation

As mentioned above, one approach which can be used to furtherconcentrate stromal precursor cells from a “spun fat” layer involves:(i) incubation with collagenase, followed by (ii) a secondcentrifugation step.

However, it must also be understood and recognized, from the outset,that IF a cell preparation is contacted by a biologically active enzyme,prior to injection of the cells back into a human, major questions aboutsafety and efficacy will arise, including questions about each and allof the following:

(i) the purity, safety, and effects of any collagenase enzyme that isused, especially if it is a non-human and/or genetically engineeredpreparation;

(ii) the presence of any residual levels of unremoved collagenase enzymethat may be present, in the treated cell preparation that is injectedinto the patient; and,

(iii) any effects which the collagenase treatment may have had on thetreated cells, as distinct from effects on extra-cellular material anddebris.

Since those types of questions will be unavoidable if a collagenasetreatment of a “spun fat” material is used, the laws that apply in theUS (and all other developed countries) would inevitable require thatextensive clinical trials must be performed, to prove to thesatisfaction of the US Food and Drug Administration (or any similaragency, in any other country) that any cell preparation treated byenzymes or other biologically active chemicals will not createsubstantial risks of adverse long-term side effects.

Those types of issues, concerns, and problems can be avoided entirely,if purely mechanical processing is carried out, using equipment which iseither disposable or autoclavable to guarantee sterility.

In view of those factors, “mechanical treatment” options, as describedin more detail below, are regarded as strongly preferable to the typesof “enzymatic digestion” option that is described in the remainder ofthis subsection.

Collagenase is a mammalian enzyme, which will cleave and break apartcollagen fibers. In soft tissues, it is always active, and is part of aconstant process of clearing out and removing old collagen fibers (whichgradually become degraded, over a span of months, causing their strengthand flexibility to become impaired), and replacing them with newcollagen fibers, which constantly are being generated and secreted byvarious types of cells. The constant process of digesting and removingold collagen fibers, and replacing them with new collagen fibers, isdirectly analogous to the process of apoptosis (programmed cell death)and cell replacement, as described in the Background section.

Accordingly, if collagenase is used to digest the extra-cellular fibrousmatrix that will be present but partially broken apart, in fluidizedfatty tissues obtained by liposuction, the collagenase treatment canindeed help release the valuable cells from a relatively sticky andclingy extra-cellular material which will not provide any benefits to apatient (and which is likely to interfere with and impede the desiredresults, in most cases) by the time a liposuction fluid has beenconverted into “spun fat”. Accordingly, a collagenase incubation step,followed by a second round of centrifugation, can theoretically behelpful, and may even be practical and useful, in some situations, forconverting a mass of sticky “spun fat” into an more concentratedpreparation which will contain substantially larger numbers and higherdensities of stromal precursor cells, per volume.

If collagenase treatment is used, it can use commercially availablecollagenase, which usually is shipped and stored in dry powdered form. Alow-viscosity aqueous liquid should be added to the incubation mixture,at a suitable volume, to function as a solvent and/or suspension agent.The total aqueous liquid in the mixture should be about half of the spunfat volume, and it should be noted that this fractional volume can andpreferably should include the quantity of aqueous liquid that was usedto wash cells off of the cell filter(s), as described above. Candidateaqueous liquids that can help promote the collagenase digestion stepinclude a buffered salt solution known as Hank's balanced salt solution(HBSS), which preferably should not contain phenol red or any otherindicator-type chemicals. If desired, a small quantity of glucose orfructose diphosphate can be added to the aqueous dilution liquid, toprovide the cells with a source of energy, to help them survive thestresses that are imposed on them while they are outside the patient'sbody. The incubation period should utilize periodic shaking, to promotemixing (such as for 10 to 15 seconds, every 5 to 15 minutes), and theliquid mixture also can be stirred or rocked continuously. Incubationshould be carried out at a temperature somewhere between about 37° C.and about 40° C., for a sufficient time to enable the enzyme to exertits effects, such as 1 hour.

After the collagenase incubation step has been completed, a secondcentrifugation step should be performed. Since a substantial amount ofdebris (including glycogen, collagen remnants, and various other typesof cellular and extra-cellular debris) will be present, the digestionmixture preferably should be mixed with a substantial or large volume ofa cell-free liquid, for the second centrifugation. Bovine fetal serum,and autologous platelet-poor plasma (which can be obtained from thepatient's own blood, as a byproduct from the platelet-rich plasmapreparation step), have been used by the Applicant herein, with goodresults, and other candidate liquids (including buffered salt solutions)can also be evaluated for use in this particular step, if desired, usingno more than routine experimentation, where (i) cell viability levels,and (ii) cell density or concentration levels, after all processing hasbeen completed, are the crucial criteria that must be evaluated andcompared.

A volume of aqueous diluent (which will effectively perform as a washingliquid, during the second centrifugation step) that can range from about25% up to about 90% of the total volume of the diluted mixture, can beused. This volume of liquid is not crucial, since the added aqueousliquid will simply be removed again by the centrifugation step. However,a low-viscosity aqueous liquid can facilitate and enhance the secondcentrifugation step, by reducing the tendency of collagenase and/ordebris to interfere with the cells, as the cells (which have the highestdensity) are driven toward the outer tip of the tube, cone, or othercontainer that holds the cell preparation during centrifugation.

Because of the volumes that will be involved, any cartridges that willbe used for this centrifugation step preferably should not be subdividedinto chambers, syringe wells, etc.; instead, the entire interior volumeof any such cartridge should be “usable”. It generally is preferable toprovide a cone-shaped “bottom” for these types of centrifuge cartridges,to cause the pellets to be compacted into a “pellet” with a reducedsize, rather than being distributed as a layer across the entire bottomof a flat-floored cartridge.

Accordingly, after centrifugation is carried out at a speed which willnot damage the cells (such as at 40 G), for a sufficient time to achieveseparation (which presumably will be about 8 to 10 minutes, in mostcases), the supernatant can be discarded, and a compacted cell pellet orlayer will be ready to be mixed with platelet-rich plasma (PRP), forinjection into the patient.

Alternately, if desired, the cell pellet can be stored in a freezer forsome span of time. The maximal limits for storage, without damaging thecells, have not been tested; however, cell storage for up to six months,at −80° C., has been tested, and no significant damage to ordeterioration of the cells was detectable, when analyzed by stainingmethods (using reagents that will permeate through non-intact cellmembranes, but not through the membranes of healthy and viable cells)that are designed to detect nonviable cells,

It should be noted that certain claims contain limitations which referto, “removing at least a substantial portion of the supernatant, fromthe layer or pellet of cells.” That phrase is intended to reflect thesimple fact that it normally does not require additional steps, or ahigh degree of care or precision, to effectively isolate a layer orpellet of cells from a supernatant, or to treat any centrifuged fluid ina manner that exploits precise boundaries between the layers. Exact andprecise boundaries, between different layers of a biological fluid,usually are not created when a centrifugation process must be kept torelatively low speeds and limited times in order to protect theviability of living cells. Therefore, when a layered fluid generated bya centrifugation process is being decanted, siphoned, pipetted, orotherwise processed to separate the layers, the typical practice in mostclinical settings is to isolate the layer of interest, plus relativelysmall “transitional layers” above (and in some cases below) the layer ofinterest. Since this practice is not exact and precise, the best way todescribe it, in language suited for a patent claim, is to simply assertthat “at least a substantial portion” of the unwanted layer(s) areremoved, from the layer or pellet of cells which are being isolated.

Mechanical Processing, to Generate Shearing Forces that Will DetachViable Cells from Extra-Cellular Collagen and Fat

As mentioned above, if a biologically active compound (such ascollagenase, an enzyme) is used to treat cells which will later beinjected back into a human, major questions concerning safety andefficacy will arise. Such questions are likely to require extensiveclinical trials, to generate statistical data that will be sufficient toprove, to the satisfaction of regulatory experts and agencies, that anysuch treated cell preparations remain safe and effective, after thebiochemical treatment.

By contrast, if mechanical processing is used, in which the cells arecontacted during such processing only by reliably sterile surfaces(which can be ensured by means of proper clinical procedures, usingdevices that are either disposable or autoclavable), the most difficultand problematic questions can be simply and cleanly avoided, and astrong presumption will arise that extensive clinical trials will not berequired, since the cells themselves will not be altered in any way,except by gently prying them away from collagen fibers in theextracellular debris.

Finally, it should be noted that if mechanical-only cell separating isdone, the only truly necessary quality control involves simply checkinga small sample of the cells, in the final pellet or layer of cells,using a device such as a light microscope and a suitable stainingreagent, to ensure that the large majority of the cells remain intactand viable, and have not been ruptured, lysed (i.e., broken) orotherwise killed or damaged.

Accordingly, FIGS. 3-5 are “rough schematic” drawings intended toillustrate several types of candidate extrusion and/or shearingmechanisms which can be used to separate viable stromal precursor cells,from the extracellular debris that will be present in “spun fat” createdby centrifuging a liposuction extract. In addition to the three “rough”schematics in FIGS. 3-5, which are intended to show candidate mechanismsrather than detailed designs, FIG. 6 illustrates, in more detail, apreferred design for a mechanical device for separating stromalprecursor cells from the extracellular matrix that is present in “spunfat”.

In any of these types of devices, spun fat is mixed and diluted with asuitable buffered watery liquid (such as HBSS, mentioned above), beforeit is loaded into a shearing and/or extrusion device. This will renderthe spun fat less viscous, and help make it easier for the cells toseparate and disengage from the relatively sticky extracellularmaterials in the spun fat. If desired, a compound such as lecithin (arelatively gentle surfactant mixture, which is already approved forcontacting and treating cells that will be returned to a patient's body)also can be added to that mixture.

Turning to the candidate mechanism shown in FIG. 3, the spun fatpreparation (preferably diluted with a thin aqueous buffer) is loadedinto the top of separator device 200, which uses a combination ofextrusion, and stirring, to generate shearing forces that will help prycells lose from the extracellular collagen fibers. The main processingcontainer 220 generally has a cylindrical shape, and can be made of atransparent material that can be sterilized in an autoclave after eachuse (such as clear polycarbonate). Inlet device 210 is positioned on topof the main chamber 220, so that gravity will assist in the flow andseparation process. The inlet device should be provided with apressure-tight inlet connector 212, such as a threaded “luerlock”fitting that will accommodate a syringe having a luerlock outlet.

The cell-plus-fat mixture is forced, by fluid pressure applied by theoperator (such as by pushing a syringe plunger into a syringe barrel) topass through an extruder or distributor plate 212, which can be made ofone or more layers of woven screen material, or which can be a flatplate having multiple relatively small holes passing through it. Earlytests indicated that extruder holes having a semi-conical shape, with aninlet diameter of 3 mm (on the top surface of the plate) and an outletdiameter of 2 mm (on the bottom surface of the plate), passing through aplate having a suitable thickness (such as 3 to 10 mm), provide goodresults for an initial extrusion step which will help getstirring/shearing activity in the main chamber 220 to function moreefficiently.

After the diluted cell-and-fat mixture enters the main cylindricalchamber 220, it will begin to rotate, as indicated by the arrows, drivenby a powered stirring mechanism 222. A conventional magnetic stirringrod 222 is illustrated, for simplicity; if desired, a more elaborate andpowerful device can be used, such as a device having two or morevertical stirring paddles attached to a magnetically-drivable base.

As the watery-fatty cell suspension circles through the chamber 220, itwill pass through a set of perforated “catch plates” 224. Because ofsurface tension factors, globules and droplets of fat and oil that aresuspended in the moving watery solution will stick and cling to thecatch plates, whenever they contact a surface of one of those plates.This will help “clear out” the mixture, and make it easier for the cellsto be separated cleanly.

For simplicity of illustration, only one catch plate 224 is shown, inFIG. 3. In early testing, a set of 3 catch plates, mounted radially at120 degree intervals around the outer wall of chamber 220, provided goodresults.

The base unit 230 preferably should be provided with a heating element,to help ensure that the suspension remains at a preferred temperatureduring the entire cell separating process. A temperature range of about105° Fahrenheit (about 40 to 41° Celsius), up to about 110° F. (about43° C.), will emulate a fever which would be very severe or permanentlydamaging (especially for brain and spinal tissue), in a living human(especially for brain tissue); however, it will not kill the types ofstromal precursor cells of interest herein, if limited to less than 10minutes. Therefore, recommended temperature ranges for optimizationtesting as disclosed herein, using any particular type of extrusionprocessing that has been selected by a specific research team, begin atabout 103° F. and range up to about 115° F. (i.e., about 40 to 46° C.).Even higher temperatures can and should be tested, by researchers whocan limit the duration of their elevated-temperature extrusionprocessing to shorter time periods.

Over a span of time, which in early tests has ranged from about 5 toabout 20 minutes, the freed and released cells will descend to thebottom floor 240 of chamber 220. Bottom floor 240 preferably should havea sloping surface, to help convey the cells to a valved outlet port 242.If the walls of the chamber 220 are made of transparent material, theperson running the separation process can monitor it visually, both toensure that everything is moving and proceeding properly inside thechamber, and to determine when the liquid reaches a state of sufficientclarity and transparency to indicate that the large majority of thecells, initially suspended in the solution, have dropped out of solutionand are ready to be removed from the chamber.

If desired, other types of treatments (which most commonly includeincreased salt content, increased acidity, etc.) which conventionallyare used to release affinity-bound molecules from sorbent materialsduring affinity purification procedures, can also be tested, todetermine their ability to help detach and separate the desirablestromal precursor cells, from the unwanted extra-cellular debris(including collagen fibers, glycogen, etc.) that is present in a “spunfat” mixture.

Passage of Spun Fat Through Screen-Type Layers

FIG. 4 provides a simplified depiction of an alternate type ofmechanical processing which can be used to detach viable stromalprecursor cells from extra-cellular debris in spun fat. In this type ofsystem, the spun fat can be passed (driven by gentle pressure) throughone or more vibrating or “tapped” screens or other permeable layers.

As used herein, the term “screen” refers to a layer of material that ismade by weaving together (or otherwise assembling, such as by knitting,affixing within clamp layers or devices, etc.) a plurality of strands(which might also be called fibers, wires, threads, or similar terms,depending on what material they are made from) into a generally flatlayer. Unless the strands are made of thick and/or relatively rigidmaterial which can transmit compressive forces, a segment of screenusually will need to be secured inside a frame, bracket, or holdercomponent, to be useful as described herein.

By contrast, the term “extrusion device (or layer)” as used hereinrefers to a device which is relatively rigid, and which can readilytransmit vibrational-type motions throughout the entire device. Suchdevices can be made by a process such as: (i) creating a plurality ofholes (or slots, openings, passageways, or similar terms, asappropriate) through a relatively flat layer of solid material (such asa plastic or polymer, or an autoclavable metal) by a suitable means suchas drilling, punching, laser cutting, etc.; or, (ii) using molding orsimilar means to manufacture a relatively flat layer of material whichhas an array of openings (or holes, passageways, etc.) passing throughthe material. In general, a regularly-spaced array of holes ispreferable to a random pattern, for use as described herein.Furthermore, any such holes or passageways can be provided with taperedsem-conical shapes, to help gently pry the cells loose from the collagenfibers, as the spun fat material is gently forced through thepassageways of an extruder device.

The distinction between “screens” and “extruders” is not precise, anddevices can be created which are at a halfway point between those twoclasses of devices. If that situation arises, either term can beapplied, depending on which term makes more sense when applied to aparticular device.

Regardless of whether a screen device (inside a frame) or an extrusiondevice is used, replaceable cartridge-type devices can be created, witheach “cartridge” containing a screen segment or an extrusion device,embedded in a somewhat thicker frame or border made of a flexible rubberor polymer. The slightly thicker frame component can create awater-tight seal when the screen or extruder cartridge is pressed into acorresponding slot which will position it in a desired location within aflow conduit or channel. Regardless of whether screen-type or extruderdevices (or some combination of both types), two or more such devices,in sequence, can be used if desired, and the two components can havedifferent design features, such as different hole sizes and/or spacing.In addition, passage of spun fat through an initial screen or extruderwhich has larger holes, and which therefore will effectively act as adistributor device, to create a more even linear flow of the thick andviscous material through the device, thereby minimizing any localizedhigher-pressure zones within the spun fat, can also be helpful.

Initial testing by the Applicant herein indicated that passage of a spunfat layer through a first screen or extruder having moderately largeopenings, (such as between 0.8 and 1.5 mm in width or diameter, whensquare holes in a screen or round holes in an extruder are being used),and then through a second screen having smaller openings (such as about0.5 mm width or diameter), can enable better cell separation andpurification, compared to passage through only a single screen orextruder.

However, it also should be noted that every passage of the cells,through a flow obstacle such as a screen or extruder, will inevitablyimpose at least some level of stress on the cells that are beingtreated. Therefore, keeping the number of such passages to a minimumwill help reduce cell mortality. Accordingly, unless and until data fromoptimization testing indicates otherwise, it is presumed that passagingof spun fat through an initial “distributor” device having moderatelylarge holes, followed by two (and only two) screens or extruder deviceswhich are mis-aligned relative to each other (so that any given cellwill need to travel through a non-linear path, to help promote betterseparation), can help achieve a good balance between maximal cellseparation, and minimal cell damage.

If a testing program is commenced to determine truly optimal (ratherthan merely adequate) design and operating parameters for the use ofscreen-passaging to separate stromal cells from extra-cellular debris in“spun fat” from a liposuction extract, each of the following parameterswill merit serious consideration, for use as a controllable variablewhich can be evaluated to determine the optimal dimensions formaximizing cell separation, while minimizing cell damage or mortality:

1. optimal hole sizes, for both (i) a single screen, and (ii) eachscreen, if two or more screens will be used;

2. optimal hole shapes. If an “orthogonal” weaving pattern is used, itwill create square or rectangular holes, with corners that nominallyhave consistently 90 degree angles. More complex weaving patterns can beused, if desired, which can create, for example, a honeycomb patternwith hexagonal holes which do not have the relatively “sharp” corners ofsquare or rectangular holes.

It should also be noted that the “nominal” 90 degree angles of thecorners of square or rectangular holes, and the “nominal” 120 degreeangles of hexagonal hole junctures, do not accurately indicate what willbe actually encountered by cells that are passing through a screen. Eventhough the “nominal” angles of the square holes in a conventional screenare 90 degrees, the actual angles which will be created, by theinterweaving of cylindrical strands which typically will have diametersat least 10 to 20 times greater than a cell diameter, will be much morecomplex, with a number of small acute wedge-shaped angles at each andevery intersection of two strands in the screening material. Thoseminiature acute angles are likely to help promote better cellseparation, even at slow cell-travel speeds, when a screen is used asdescribed herein, and their effects on cell damage and mortality arelikely to increase at increasing rates (possibly even approachingexponential increases), as cell travel speeds are increased. Therefore,a range of cell travel speeds (which can be controlled and modulated, bycontrolling pressure gradients and pumping rates) will need to beevaluated, when any particular screen dimensions and weaving pattern arebeing evaluated.

Alternately, those types of factors and issues can be avoided (or atleast shifted to lower levels of significance) by: (i) creating a layerof plastic, polymer, metal, or similar material, and then (ii) punching,drilling, laser-cutting, or otherwise treating that sheet of material,to create an array of holes through the sheet. The holes can have anydesired size, and any desired pattern (such as square grids, or“honeycomb” patterns). However, it must be noted that whenever amachining process is used on a pre-existing sheet of material, it maycreate a set of “directional” traits in the treated sheet. For example,if a punching or drilling process is used, the “top” side tends to haveslightly rounded depressions surrounding each hole, while the “bottom”side of each hole is likely to be surrounded by small irregular spurs orfragments of material, protruding outwardly from the main sheet.Accordingly, if a screen is created by machining an already-formed sheetof material, the “direction of machining” should be recorded, andtested, as a potentially significant factor when the screen is used forseparating cells from debris, in spun fat.

3. If a cell-separation screen is made from woven strands, the strandthickness(es) become a design and operating parameter which can becontrolled, and which should be tested and optimized. If the strands aretoo thin, they can cut open and kill cells, in a manner comparable to acheese slicer which uses a thin wire to cut through a block of cheese.This concern is aggravated by the fact that the stromal precursor cells,in a spun fat suspension, will not be surrounded by a watery liquidwhich will allow the cells to simply move aside and flow around anobstacle they encounter; instead, the cells will be surrounded by, andeffectively embedded within, a thick, sticky, viscous mass of fattysemi-solid material from which most of the water and oil has alreadybeen removed, rendering the “spun fat” even thicker and more viscous andsticky than normal fat.

Strand diameters, in a material used to make a cell-separating screen asdescribed herein, can be expressed as a multiple of the cell diameters.Because of the factors discussed above, a presumption arises that: (1)the strands should be made from monofilaments (either polymers, or metalwires) with smooth cylindrical surfaces, rather than from woven orbraided strands of even smaller fibers; and, a good starting thickness,for optimization testing, can be provided by strands which havethicknesses (diameters) that are about 20 times greater than therelevant cell diameters. Since the diameter of a typical non-fibrouseukaryotic cell is roughly 10 microns (i.e., 1/100 of a millimeter),this implies that a good starting thickness, for optimization testing ofstrands that will be used to make cell-separation screens, will be in arange of about 200 microns, or 0.2 mm.

4. The number of screens, the linear distance which separates thescreens, the “linear flow” rates and speed of cell travel through thescreening conduit, and the pressure gradient between the inlet and theoutlet of a screen-passaging chamber, should all be treated ascontrollable design parameters, in any optimization testing.

5. The ability to impart vibrational, reciprocating, or other motion, toany or all of the screens, also must be considered as a testing anddesign parameter that can be controlled and evaluated. As describedabove in relation to extruder devices, such motion can be asinusoidal-type vibration, or a tapping, hammering, or other jarringmotion. In either case, frequency and amplitude can each be adjusted,and then evaluated.

6. In addition, a screen or extruder can be vibrated or tapped in avariety of patterns, such as: (i) a single back-and-forth linear motion;(ii) a rotary or elliptical motion; or, (iii) a motion which alternatesbetween two or more different directions, such as alternating up/downand left/right vibrating or tapping.

7. In addition, various known methods can be used to introduce any oneor more of several different types of energy inputs (such as sonic orultrasonic waves, physical vibrations, or electrical fields) into a cellsuspension that is being passed through one or more screen-separators.Similarly, a stirring paddle or similar device can be used to stir oragitate the cell suspension, or to otherwise subject a fat-and-cellmixture to shearing forces just before, or just after, the cells passthrough a screen or extruder.

Shearing-Force Agitation of a Spun Fat Material

FIG. 5 is a simplified depiction of a fluid-handling system which willforce a spun fat preparation through a conduit in which rotating,reciprocating, or otherwise moving paddles (which can also be calledagitators, blades, or similar terms) are used to generate shearingforces that will help detach cells from extra-cellular collagen fibersand debris.

FIG. 5 depicts two sets of paddles, each mounted on a central verticalaxle (not shown), which are angled and which are rotating in differentdirections, to generate shearing forces in the turbulence they willcreate. Any number of such rotating paddles can be used, and they do notall need to be mounted on a single centered axle; for example, a paddlesystem can be designed with effectively interlocking “fingers” whichwill “sweep” across an enclosed area, in which the “even-numbered” and“odd-numbered” paddles will be moving in opposite directions at nearlyall times, except when they pause to change directions. These arematters of relatively straightforward design, and they can usesophisticated stirring systems which already have developed for eitheror both of two fairly common uses: (i) manufacturing of liquid mixtureswhich contain ingredients that do not mix well together naturally, butwhich must be mixed together very thoroughly, to ensure consistentquality of the complete mixture; and, (ii) storage of liquids that needto be kept mixed together thoroughly.

If those types of devices are used to generate shearing forces that willhelp detach stromal cells from extra-cellular debris in a spun fatmaterial, the design and operating parameters which should be evaluated,during optimization testing, including the following:

1. The dimensions and other traits of the paddles which will be used,including their overall shape, their thickness, the shapes and contoursof their edges, and the presence, size, spacing, and contours of anyholes that are present in the paddle surfaces;

2. The slanted angles, rotational speeds, spacing, and placement of thepaddles, within a stirring chamber;

3. The average linear speed of the cell suspension which is travelingthrough the stirring chamber; and,

4. whether (and to what extent) any additional energy or other inputs(such as sonic or ultrasonic waves, vibrational motion, electric fields,etc.) will also be introduced into the stirring chamber while the cellspass through it.

Extrusion Device which Uses Balloon Catheter

As noted above, FIGS. 3-5 are “rough schematics” which are intended toillustrate several candidate agitating and/or shearing mechanisms whichcan gently pry cells away from the extracellular debris in a “spun fat”mixture.

By contrast, the two drawings in FIGS. 6 and 7 depict a specific designwhich will be used as the basis for designing, fabricating, and testingprototypes of a system which, as this is being written, is preferred bythe Applicant for expanded testing and evaluation.

As a brief overview, the main components of cell concentrator 300 areshown in FIG. 6, while FIG. 7 shows the same type of cell concentrator300 with an additional component that can help increase cell viability.

In FIG. 6, cell concentrator 300 is enclosed within an outer shell 310,which has dimensions that will allow it to fit into a holding cartridgein a desktop centrifuge, of the type used to prepare “platelet richplasma” (PRP). As described in the Background section, most modernclinics and offices that specialize in “sports medicine” and/ororthopedics work will already have a PRP centrifuge, on site, sinceplatelet cells (which can be obtained from a sample of blood from thesame patient who needs repair work) are heavily and actively involved intissue repair. Accordingly, cell concentrator 300 is designed to fitinto a centrifuge cartridge that will fit into a conventional type ofPRP centrifuge.

As an additional prefatory comment, cell concentrator 300 is designed toenable all necessary steps to be carried out with a high level ofsterility. These devices will be manufactured and then packaged insealed sterile envelopes, to be used once (as a disposable component)during an operation on a specific patient, and then discarded as medicalwaste. Although the inlet and outlet fittings are not shown in detail onFIGS. 6 and 7, the inlet port 322 for the spun fat, and the outlet port314 for extruded cells, will be provided with sterile fittings whichwill allow secure attachment of a tube or syringe device, such asconventional “luer”-type (or “luerlock”) fittings. Those inlet andoutlet attachment components will be covered and protected by caps thatare left in place until immediately before a connection is made. If aclinic has a “sterile hood” or “glove box” available, those can be usedto handle and manipulate these devices with even higher levels ofassured sterility; however, hoods and glove boxes are large andexpensive, and most clinics that specialize in sports medicine do nothave or use them, so the cell concentrator devices disclosed herein aredesigned to not require them.

As a feature which illustrates that comment, the main outer shell 310has an accommodating cap 320 which will be affixed to it (such as bythreads, slots, or glue), during manufacture. This will enablepositioning and securing of the internal components inside the maincylinder, while the top end of the main shell 310 is open andaccessible, during manufacture. Once the cap 320 has been secured to themain shell 310, the assembled device 300 will be sealed inside a sterileenvelope and shipped to a physician's office or clinic, and the devicewill be used by the physician without ever removing cap 320 from mainshell 310.

Cap 320 has a first inlet 322 (which also can be called an orifice,opening, passageway, channel, etc.) for receiving spun fat that is beingforcibly expelled out of a syringe barrel that has been spun in acentrifuge. Inlet 322 (which is provided with a luer-type or similarfitting, to allow a secure water-tight attachment to be made to it) willcarry the spun fat to the top opening of an extrusion cylinder 330(discussed below), located inside the outer shell 310 of cellconcentrator 300. Inlet 322 presumably should be centered, with acone-shaped conduit which carries the spun fat to distributor plate 324(the conduit channel is indicated by dotted lines; solid lines are usedto show most internal components, since these devices very likely willbe made of clear and transparent plastic or polycarbonate material, toallow the physician to visually monitor the progress of the loading,extrusion, and centrifugation steps described below).

Distributor plate 324 has relatively large holes passing through it. Itcan be molded into (or otherwise affixed to) either the cap 320, or thetop of extrusion cylinder 330 (discussed below); either way, the goaland purpose of distributor plate 324 is to load and distribute the spunfat evenly, around an “inner annular gap” 331 which surrounds anelongated balloon catheter 340, positioned in the center of extrusioncylinder 330.

Cap 320 also has a second conduit 326 (also with a luer-type or similarattachment fitting) passing in a generally vertical direction throughcap 320. A flexible tube 328 will be coupled to the bottom outlet ofconduit 326, and will carry a pressurized fluid from aphysician-controlled pump, compressor, or other actuator, into ballooncatheter 340, discussed below.

Moving down in FIG. 6, extrusion cylinder 330 has a number of relativelysmall extrusion holes 332 passing through its cylindrical wall. In FIG.6, these are represented by several rows of extrusion holes, shownflanking the balloon catheter 340 so that they will not interfere withthe view of that balloon catheter. In an actual working unit, there willbe a fairly large number of extrusion holes, such as at least about 50,and up to about 500 holes, evenly spaced around the circumference andlength (or at least most of the length; bands around the top and bottomregions might not have holes) of extrusion cylinder 330. Alternately,cylinder 330 can be made from a segment of screen, woven from fibers ofsuitable material and diameters, with suitable spacing.

In FIG. 6, the bottom surface 334 of cylinder 330 is shown as beingsolid. However, it can be perforated if desired, or it can be formedfrom screen material, in a manner comparable to the toe of a sock.

After spun fat has been loaded into the inner annular gap 331, betweenextrusion cylinder 330 and balloon catheter 340, the extrusion processwill be driven and controlled by forcibly injecting a compression fluid(such as air, an inert gas, or a liquid such as buffered salinesolution) into the balloon catheter 340. Catheter 340 has two stiffmounting rings 342 and 344, surrounding its top and bottom ends, whichprovide a means for a plurality of radial struts 345 to hold thecatheter 340 in position, aligned lengthways along the main axis in thecenter of extrusion cylinder 330.

A flexible, expandable, inflatable polymeric membrane 346, generally inthe shape of a flattened tube when not inflated, extends the length ofthe balloon catheter 340, between upper and lower mounting rings 342 and344. When air, an inert gas, or a liquid is forcibly driven into theinflatable membrane 346, that membrane will expand, thereby enlargingthe volume of the balloon. This will forcibly displace the spun fat outof the inner annular gap 331, through the extrusion holes 332, and intothe outer annular gap 311. Preferably, the balloon should be designedand sized so that its volume, when inflated, can occupy essentially theentire volume of extrusion cylinder 330, with the side walls of theballoon pressing directly against the interior wall of the extrusioncylinder.

Balloon catheter devices are well-known, and have been developedextensively, and used for decades, for medical uses such asminimally-invasive manipulation of clogged arteries. In a typical bloodvessel procedure, a balloon catheter having an appropriate diameter andlength is “snaked” (using a long hollow tube as the handle) into thesite of a fatty, cholesterol-laden “plaque” that is clogging or blockingan artery. The balloon is then inflated, by forcibly pumping a smallquantity of a non-compressible, non-toxic liquid (such as a bufferedand/or supplemented saline solution) through the hollow tube and intothe inflatable balloon. As the balloon expands, it forcibly compressesand flattens the “plaque” that was clogging the artery, thereby openinga larger channel for blood flow through the plaque. In most cases, anexpandable mesh device called a “stent” is then inserted into thenewly-opened channel which passes through the flattened plaque, and leftin that position as the catheter is withdrawn, to help ensure that theclogging problem will not recur (at least, not at that location) for atleast several years.

As mentioned above, balloon catheters are well-known, and have been usedfor decades. Illustrations and descriptions can be easily found on thewebsites of surgical supply companies which manufacture and sell them,such as, for example, the websites of Boston Scientific, Medtronics,Spectranetics, and Teleflex Medical.

In addition, any company which sells balloon catheters will also sell atleast one type of inflator control device which it recommends, for usewith its inflatable catheters. One example is the ENCORE™ inflatordevice sold by Boston Scientific. As described and illustrated atwww.bostonscientific.com/en-US/products/accessories/encore.html, thisdevice allows a physician to forcibly inflate and expand a ballooncatheter by manually rotating a handle at one end of a large-diameterthreaded shaft. As that shaft is forcibly screwed into an accommodatingliquid-tight cylinder, the travel of that shaft, into the cylinder,forcibly drives a noncompressible liquid (such as aqueous salinesolution) out of the opposing end of the cylinder, and into the ballooncatheter. Therefore, the ability to rotate a handle attached to athreaded shaft provides a trained physician with a good sense of “feel”for what is happening inside an artery, as the catheter expands. Thatsense of “feel” is further enhanced by the physician being able to watcha live image of the balloon catheter as it inflates and expands, usingvideo images that are similar to X-rays, on machine called afluoroscope. This allows the physician to see and monitor the progressof the catheter expansion, as he rotates the handle which drives thedisplacing shaft deeper into the pump cylinder.

That level of control and “feel”, which is very useful for helpingprevent damage to a fragile artery which is being manipulated andaltered inside a patient, will not be necessary for pressing “spun fat”through an extrusion cylinder in a beaker or similar container. Instead,a consistent and known level of pressure (which can be made to increasegradually at the start of the extrusion process, and gradually taper offas the final inflation level is approached) is likely to be preferred,whenever a particular combination of balloon catheter and extrusioncylinder are being used (that optimal pressure, for any particulardevice, will depend on factors such as the number and sizes of theextrusion holes, in the extrusion cylinder or screen). In addition, tohelp minimize abrupt motions which might create shear or other forceswhich could damage the cells, a presumption arises that an optimal“plateau” pressure for inflating a balloon catheter and extruding cellsshould be approached gently and gradually, at the start of the extrusionprocess, and should gradually be tapered off, as the final inflationvolume is approached.

Alternately or additionally, a quick and simple visual viscosity test(which can involve, for example, watching to see how many seconds itwill take for a standardized metal rod to descend downward, through asample of spun fat from a specific patient) can be used to determine theoptimal pressure level for extruding a batch of spun fat from anyparticular patient.

Accordingly, by using the type of device shown in FIG. 6, a “spun fat”preparation, containing stromal precursor cells clinging to a stickyextra-cellular matrix of unwanted debris, can be forcibly extrudedthrough a large number of small holes, in a manner which providesmaximal cell separation, with minimal cell damage. As shown by thedirectional arrows in FIG. 6, the extruded material will be forced outof the “inner annulus” 331, inside extrusion cylinder 330, into the“collection annulus” 311, located between extrusion cylinder 330 andouter shell 310. As the extruded cells, collagen fibers, fat, oil, anddebris pass through the holes of extrusion cylinder 330, they will falldownward, pulled by gravity, toward the bottom of outer shell 310, whichhas a tapered lower end 312 which leads to an outlet port, which willhave an attachment component (such as a luer or luerlock fitting), whichwill be covered and sealed by a cap during the extrusion andcentrifuging steps.

A complete extrusion process is likely to involve two or more cycles of:(1) loading a volume of spun fat into the extrusion cylinder; (2)forcing the fat out of the cylinder, by inflating the balloon; and, (3)deflating the balloon, to make room for another batch of spun fat.

When the desired number of cycles has been completed and the collectionannulus 311 is suitably full, the spun fat supply conduit, and theconnecting tube used to inflate the balloon catheter 340, will bedetached from inlets 322 and 326, and the cell concentrator 300(containing the extrusion mixture) will be loaded into a centrifugecartridge. A centrifuging step, carried out at a speed and durationwhich will not damage the cells (as discussed above) will cause thecells (which are filled with cytoplasm, an aqueous liquid which isdenser than oil) to be driven to the lowest region of the conical tip ofcell concentrator 300, while the fat, oil, and collagen fibers will formother layers higher up in the tube.

A highly enriched cell population can then be removed from theconcentrator 300, by forcing or suctioning them out of the outlet 314 atthe bottom of conical floor 312 of device 300. This enables theconcentrated cells to be subsequently injected back into the patient,while leaving the fat, oil, and collagen fibers in the collectionannulus 311 of cell concentrator 300, to be discarded as medical waste.

It is possible to use a gas, such as ambient or filtered air, ornitrogen, to inflate the balloon, since the pressurized fluid willremain inside the balloon catheter, and will never contact the spun fator cells. However, not everything works perfectly every time, andsubstantially less damage to the cells will be caused, if a balloonfilled with an aqueous liquid (such as buffered saline, or a mixturesuch as Ringer's lactate or Hank's balanced salt solution) ruptures,compared to a balloon filled with compressed gas. When a gas iscompressed, the reduction in volume becomes a form of energy storage,and if a balloon filled with compressed gas suddenly pops, that pent-upenergy will create a concussive-type shock wave that will spread outwardin all direction, killing cells in the near vicinity, and damaging cellsfarther away. By contrast, water is essentially incompressible,volumetrically; therefore, if a leak forms in a water-filled balloon,any stored energy will dissipate almost immediately, usually with asingle small, targeted, limited spurt of water. That type of “water jet”may kill a few cells in the direct path of the jet, but it will notcreate a much more damaging concussive shock wave that spreads out inall directions.

Furthermore, the ability of gases to change volume, when compressed,creates a type of “disconnect” (or “uncoupling”, or similar terms)between a controlled pumping action, and the result of that action. Aphysician's ability to “feel” what he is doing, and to reliably knowwhat is happening as a direct result of what he is doing, would beimpaired, by the use of compressible gas in a balloon catheter forforcing spun fat through an extrusion device. Using a compressible gasto inflate the balloon, in this setting, which would be analogous totrying to use a scalpel during a delicate operation which needsprecision, while wearing thick padded gloves.

Finally, use of a compressible gas, to force a sticky paste-likematerial through extrusion holes, would be more likely to cause smallhigh-velocity spurts of cell materials, each time a plug of materialwhich is clogging an extrusion channel is forced through the channel byincreasing pressure levels. Those types of high-velocity spurts can killcells, so they should be avoided and minimized, whenever possible, byusing a non-compressible aqueous solution, rather than a compressiblegas, to inflate the balloon catheter in an extrusion device as describedherein.

In view of those factors, a presumption arises that, unless actualtesting indicates otherwise, the balloon catheter should be inflated bymeans of an aqueous liquid, rather than a gas.

Testing to date has indicated that extrusion hole diameters of about 0.5mm are well-sized to promote a good balance between: (i) high levels ofcell separation from the collagen fibers, fat, oil, and other unwantedextra-cellular components and debris in “spun fat” from a liposuctionextract; and, (ii) low levels of cell damage and mortality. Rather thanstating that that size is optimal, it is regarded as a good and usefulstarting point, and the exact dimensions and material(s) for a trulyoptimal extrusion cylinder can be determined using no more than routineexperimentation, with 0.5 mm diameter holes as a good starting point foroptimization testing. Any such testing program should evaluate, for anycandidate material and operating parameters, the balance and trade-offbetween:

(i) cell separation and yield levels, which should be as high aspossible; and

(ii) cell mortality levels, which should be kept as low as possible.

In addition to evaluating hole sizes, a testing program for creatingtruly optimized materials for making extrusion cylinders for use asdescribed herein should evaluate as many of the following parameters aspossible:

(i) the type of machining process used to create the holes, such asdrilling, punching, molding, or laser cutting, as well as weaving offibrous strands made of various materials (including mono-filament,braided, and poly-filament) and diameters, into screens having variousthread diameters and weaving densities;

(ii) whether sharp edges (as created by drilling or punching), orrounded edges, will provide better results (rounded edges can be createdby, for example, passing a polymer in front of an infrared light, heatgun, or other heat source, so that it will reach a temperature highenough to soften the plastic enough for any sharp edges around theextrusion holes to melt slightly and then re-harden);

(iii) the optimal material for the extrusion cylinder (such as plastic,metallic foil, or possibly a sputter-coated, vapor-deposition, or othercoated material);

(iv) the wall thickness of the extrusion cylinder; and, if the cylinderwall is thick enough to allow shaped channels, whether the extrusionholes should have slightly or substantially conical cross-sectionalshapes, with outlet diameters smaller than the inlet diameters;

(v) the optimal pressure level for pushing and driving spun fat throughthe extruder cylinder; and,

(vi) whether the balloon inflation should be used to create an elevatedpressure inside a sealed gas-tight cell concentrator 300, or whether apressure vent or regulator should be provided, such as by attaching asmall air outlet (covered by a microfine filter to prevent microbialentry), or a pressure control valve, to inlet port 222.

The results of that type of optimization research may lead to apatentable component, device, or process; however, this type ofoptimization is not required to create a functioning and efficientsystem, which can indeed be created by following the guidelines herein.Furthermore, that type of optimization testing can be performed using nomore than ordinary skill in the art, by anyone who understands thedesired outcome of the cell separation process, and who understands howto evaluate cell separation, mortality, and concentrations (or who canhand over batches of extruded spun fat to someone else who specializesin evaluating cell damage and viability).

Additional Device to Sequester Oils from Spun Fat

During the Applicant's testing of a cell concentrator as describedabove, he discovered and recognized a significant problem which had notbeen known previously; and, he figured out how to create an enhancementcomponent which can minimize that problem, leading to higher and betterlevels of cell viability and cell yield.

The problem the Applicant recognized was that, if spun fat is passedthrough an extrusion device which is well-suited for prying cells loosefrom collagen fibers, fat, and extra-cellular debris, so that the cellscan be further concentrated, the liquefied oil and fat which arereleased, by the spun fat, becomes seriously toxic to the cells, withinthe time frame that is required by extrusion followed by centrifugation.This factor becomes even more pronounced and serious, if the extrusionprocess is performed at an elevated temperature, such as about 105° F.Mildly elevated temperatures, which emulate a serious but not fatalfever, helps soften and liquefy the fatty material, which helps releaseits grip on the cells.

Accordingly, leaving newly-separated cells in direct contact (for asustained period of time) with an oily fluid that was created by heatedextrusion of spun fat, causes a substantial die-off and loss of viablecells. The reasons for that type of cell damage and mortality have notyet been closely studied, but it is presumed that any such oil, if givensufficient time in direct contact with the cells at a highconcentration, will create an aggressively sticky coating, which willclog and foul large numbers of surface receptors which are located onthe surfaces of the cells. Proper functioning of at least most of thesurface receptor complexes, on any cell, are essential for the healthand viability of the cell. Therefore, if those surface receptor proteinsare allowed to become clogged, coated, and fouled by a thick, viscous,and sticky semi-liquefied coating, while the cells are outside the body,it is entirely reasonable (and even predictable) that that type ofsurface receptor fouling and disruption will begin to kill the affectedcells. That type of coating and fouling activity is similar to the typeof suffocation that would occur, if a human or animal had a large massof sticky oily material forced into its windpipe and lungs.

To minimize that problem, the Inventor herein developed, andsuccessfully tested (with good results), an optional additionalcomponent (i.e., an enhancing device) which can isolate, sequester, andremove oils and softened fatty materials from spun fat material, afterthe spun fat has been forced through small extrusion holes, while theentire batch of cellular material and debris remains inside the cellconcentrator, without disrupting or impeding the process.

That additional component is illustrated in FIG. 7, which shows (inslightly simplified form, to help focus upon the new additionalcomponent) the same type of cell concentrator 300 that is illustrated inFIG. 6. As in FIG. 6, cell concentrator 300 contains main cylindricalshell 310, cap 320, an extrusion cylinder 330 inside the main shell 310,and a balloon catheter 340 inside the extrusion cylinder 330. Theadditional component is an oil sequestering ring 350, shaped as anannular (ring-shaped) disc, which travels up and down (with the aid of ahandle component 351) inside the “collection annulus” 311.

Oil sequestering ring 350 comprises three layers. On its bottom surface,which will directly contact the extruded spun-fat mixture that has beenforcibly driven through the holes in the side wall of extrusion cylinder330, it has a relatively thin “lipophilic” cell filter 352. As indicatedby the term “lipo-philic” (i.e., oil-attracting), that bottom filterlayer 352 will actively “welcome and encourage” any oil and liquefiedfat, in the “extrudate” mixture, to travel upward, into and throughfilter layer 352. That oily material will be pulled upward by capillaryattraction, supplemented by downward pressure which can be exerted onthe oil sequestering ring 350 by use of the handle component 351. Thepore sizes in that thin filter layer should be small enough to block andprevent any of the stromal precursor cells from moving upward into orthrough filter layer 352. These types of filters are sold by numerouscompanies, which can be located by an internet search for “cellfilters”.

The thick center layer 354, shown in a side elevation view in FIG. 7, ismade by packing, into the outer shell of the ring device 350, asubstantial quantity of aggressively lipophilic fibers. These types ofmaterials also are well-known and readily available, and are commonlyused, in pad and roll form, for cleaning up oil spills from solid and/orwater surfaces. When oils and liquefied fats contact these types oflipophilic fibers, they will permeate and settle into those fibermasses, in a stable and comfortable manner. This will effectivelysequester the oil away from the stromal precursor cells, which cannotreach that packing material because of the cell-blocking filter layer352 on the bottom surface of ring device 350.

The top layer 356 of ring 350 can be made of any suitable solidmaterial. Its main purpose is to allow the handle component 351 to beused: (i) to move the ring device 350 upward or downward, at will,inside the collection annulus 311, even after the walls of thatcollection annulus 311 have become coated by sticky globs of material;and, (ii) to press the bottom surface of the ring device downward, withsubstantial force, into the mass of cells, oils, and debris, so thatpressurized contact with the bottom surface of ring device 350 will helpdraw oils and fatty material up into the ring, thereby minimizing theircontact with the cells, and their ability to coat, clog, and damagethose cells.

If desired, additional steps can be taken to further reduce any contactbetween: (i) the oils and fats that have been absorbed by the ring, and(ii) the stromal precursor cells that are being concentrated within thecell concentrator 300. For example, after the extrusion processing hasbeen completed, and the device is almost ready to be placed in acentrifuge, the cap which was placed on top of inlet 322 can be removed,the oil sequestering ring 350 can be lifted to its highest possiblepoint by raising handle 351, and a modest quantity of a suitable aqueoussolution, such as HBSS, can be loaded into the extrusion cylinder. Thethin and watery aqueous solution will pass readily through the extrusionholes 332, and it will form an aqueous layer that will act as a barrier,during centrifugation, between the oil-sequestering ring 350 and itsabsorbed oils, and the stromal precursor cells which are lower down inthe collection annulus 311.

Alternate Mechanisms for Driving the Extrusion Process

As can be readily understood by mechanical engineers or designers,various other mechanisms can be used to forcibly drive a spun fatmaterial through an extrusion device or screen, in a cylinder which issmall enough to be loaded into a centrifuge cartridge.

As a simple example, if an extrusion cylinder is created from a flexiblescreen, and one end of the screen is affixed to a mechanism which willforcibly roll up the screen around an elongated axle (in a mannersimilar to rolling up a window shade), that rolling-up action willdirectly decrease the volume inside the screen-cylinder, in a mannerwhich will forcibly eject the spun fat from the inside of thescreen-cylinder, out through the holes in the screen.

Another type of ejection mechanism is illustrated in FIG. 8, whichdepicts a threaded shaft 440, having an outside diameter which is almostas large as the internal diameter of the extrusion cylinder 430. Theexternal threads on shaft 440 pass through a set of accommodatingthreads which have been molded into a solid bottom floor of extrusioncylinder 430. A thin driveshaft 442, with a handle 444 at its bottomend, is reversibly affixed to the bottom of large shaft 440, so thatrotation of handle 444 will forcibly drive the large shaft 440 into theinterior of the extrusion cylinder 430. This will displace the spun fat,causing it to be forcibly driven out of the cylinder, through theextrusion holes in extrusion cylinder 430.

Use of Centrifugal Force to Drive Extrusion

If desired, the centrifugal force that is being exerted on a liquid, ina tube that is being actively spun in a centrifuge, can be used as thedriving force which will push spun fat material through the extrusionholes in an extrusion device. Two types of devices which can accomplishthat result are depicted in FIGS. 9 and 10.

FIG. 9 depicts a centrifugation cylinder 500 having an outer barrel 510,and a cap with an inlet port 522 for receiving spun fat, which will thenpass through a distributor plate 524 having relatively large holes.Before centrifugation begins, the spun fat will initially rest on top ofa cone-shaped extrusion barrier 530, which is positioned at a midpointalong the length of the outer barrel 510, and which has a large numberextrusion holes (with relatively small diameters, such as 0.5 mm)passing through it.

If this type of cylinder 500 is used, a quantity of spun fat will beloaded into the upper chamber (i.e., the volume above extrusion barrier530). The semi-loaded cylinder 500 will be centrifuged, preferablybeginning at a relatively slow speed which can be gradually increased toa maximum chosen level over a span of a minute or more. During thespinning operation, the outward-directed centrifugal force (in acylinder which has rotated out to a generally horizontal angle, duringhigh-speed centrifugation) will drive the spun fat material through theextrusion holes in the extrusion barrier 530, into a collection zone512, in a manner which will pry the stromal precursor cells loose fromcollagen fibers and other extra-cellular debris.

If desired, that type of “initial loading cycle” can be repeated, asecond and possibly a third time. Each time the majority of the “upperchamber” has been “unloaded” (i.e., by passage of the spun fat materialthrough the extrusion barrier, into collection zone 512, the “loadingstep” centrifugation can be temporarily halted, and more spun fat can beloaded into the upper chamber.

If desired, this type of system can be designed to allow an extrusionbarrier to either:

(1) float, automatically, on the top surface of the liquid that haspassed through the extrusion barrier; or,

(2) be manually repositioned, between loading cycles.

Floating-type placement can be enabled by either or both of: (i) makingthe barrier from a low-density plastic, which can be made from either aconsistent semi-foam type of material, or a layer or compartment whichcontains a low-density foam; and/or, (ii) one or more empty flotationchambers, on or near the bottom surface of the barrier.

Adjustable manual positioning can be enabled by other means, such asdesigning the barrier to have three or four extruding pins, distributedevenly around its outer edge, and providing pin-tracks with “detente”stops at various spaced intervals along the lengths of the tube.

In addition, the bottom (i.e., collection) compartment of the outerbarrel can be provided with:

(i) one or more baffle structures (such as one or more spiral-shaped orother sloped structures which have their main axis aligned with the mainaxis of the centrifugation tube);

(ii) a modest quantity of a low-density cushioning foam or liquid;and/or,

(iii) a segment of three-dimensional mesh (as described in the followingsubsection), having density and porosity traits that will provide cellswith a relatively unchallenging pathway to the bottom of the tube.

Any of those three approaches (or a combination of those types ofdevices) would ensure that cells which emerge from the extrusion holeswill not be flung a significant distance, at high speed, beforeimpacting against a hard surface, which otherwise could damage and killsubstantial numbers of cells.

A presumption applies that if viable cells are forced to cross arelatively sharp or otherwise abrasive and/or scraping edge, while underpressure, some of the cells are likely to be damaged or killed by thatmechanical stress. To minimize that effect, the extrusion holes can bemolded, laser-drilled, or otherwise formed at a “downward” angle, ratherthan having to be perpendicular to the sloping surface of the extrusionbarrier 530.

An alternate design for avoiding any “turning a difficult corner”stresses on the cells, as they pass through the extrusion holes, isshown in FIG. 10. This drawing depicts an extrusion device 630 referredto herein as a “Christmas tree” extruder, because its shape somewhatresembles a Christmas tree. To simplify and clarify the drawing, the topsurface 610 of cylinder 600 is shown as having a simple planar shape; inan actual design, it presumably will have the same type of structureshown in the alternate embodiment in FIG. 9, with an inlet port for spunfat, which will then pass through a distributor plate 620 withrelatively large holes.

The “Christmas tree” extruder 630 has a series of annular plates 632,634, and 636, as well as a bottom plate 638, all of which havesmall-diameter (e.g., 0.5 mm) extruder holes passing through them.Watertight conical walls (such as wall 633) connect the annular surfacesand the bottom plate to each other, so that no spun fat material canreach the collection zone 650, or the cell outlet port 652, withoutfirst passing through an extrusion hole in one of the annular or bottomplates.

In this manner, the cells will be driven through the extrusion holes ina direction which is fully aligned with the extrusion holes. Thatdirection is illustrated as vertically downward, in the drawing of astationary vertical cylinder 600 in FIG. 10; it will be radiallyoutward, when a loaded cylinder 600 is spinning at high speed in acentrifuge.

Centrifuging Cells Through a Fibrous Mesh/Matrix

Another approach that merits early evaluation for use as describedherein involves placing a three-dimensional fibrous mesh on top ofeither: (i) a permeable support plate, with relatively large holespassing through it; or, (ii) a loose mesh, which is designed to allowrelatively rapid, easy, low-stress travel of viable cells through thatloose mesh.

An example of such a device 700 is depicted in FIG. 11, which has anouter wall 710, a spun fat inlet port 720, a distributor plate withlarge holes 722, and an initially empty loading space 730. Loading space730 is filled with spun fat, which is then centrifuged, presumably at arelatively low starting speed which will be gradually increased, over aspan of a minute or more.

The centrifugal force will drive the spun fat through the initialfibrous mesh 740. If that mesh 740 has suitably optimized traits, thiscan “pry loose” the stromal precursor cells from the extra-cellularmaterial in the spun fat, allowing the cells to be collected, inconcentrated form, at the bottom of the tube.

In one embodiment, depicted in FIG. 11, mesh 740 is supported, withindevice 700, by a segment of a relatively loose and open low-density mesh750, which serves as both: (i) a physical support for the higher-densitymesh 740; and, (ii) a non-difficult, non-challenging pathway that willallow detached cells to weave and work their way to the bottom of thetube, for collection, rather than undergoing the risks of a high-speed“flinging” effect.

Alternate arrangements can be developed for supporting the denser mesh740; such means can use, for example, a either a fixed permeable plate,or a movable and effectively “floating” permeable support (made of alow-density foam-type plastic or polymeric material) which can beginnear the bottom of the tube, as the tube is initially filled with spunfat, and which can rise toward the top of the tube, as separated cellsand debris work their way (driven by centrifugal force) through the mesh740 and then through the holes in the floating permeable support.

Accordingly, if a “spun fat” mixture obtained from liposuction fluid isforcibly driven through one or more three-dimensional fiber matriceshaving density and porosity traits which have been optimized for thispurpose, using a high-speed centrifuge to provide the driving force, themesh will help “pry loose” stromal precursor cells from extra-cellularmaterials, in the spun fat. Accordingly, this type of design merits thetype of optimization testing disclosed herein, to determine out whethera mesh-type unit can provide separated cell viability levels which arein the vicinity of 85% (or possibly even better), which has already beenachieved by the Inventor herein using other systems as disclosed andillustrated herein.

Use of Specialized Centrifuging Beads, to Help Separate Cells

As yet another alternate approach which merits optimization testing, asdisclosed above, to determine whether it can approach or possibly evenexceed the 85% separated cell viability levels mentioned above,specialized beads having any desired density can be selected, and usedto form a “lightly packed bed” which can help separate stromal precursorcells, from collagen fibers and extracellular debris in spun fat.

The term “beads” is used herein in the same manner that refers to thetypes of very small manufactured particles or pellets which are used invarious types of biochemical separations, such as in affinity columns,chromatography columns, etc. Most of these types of beads areconventionally made from either polymer, starch, or silicone materials,and the “raw” or “core” pellets can be coated by any of numerous typesof reagents (such as monoclonal antibodies, reagents to create eitherpositive or negative ionic charges, reagents which will allow the beadsto be removed from a liquid and/or affixed to other surfaces, etc.).They typically have very small diameters, usually measured in microns,and the smallest ones are invisible to the naked eye, when seen inisolation. However, they can be manufactured in any desired diameter, upto several millimeters, which is well up into the easily visible range.

Accordingly, FIG. 12 depicts a centrifuge tube 800, with an inlet port820 for spun fat, and a distributor plate 822 leading to a loading space830. A mass of small beads 840 is contained in the bottom of the tube,above outlet port 850.

If this design is used, the spun fat will be loaded on top of the massof lightweight, low-density beads, inside a tube which is thencentrifuged at high speed. During centrifugation, those two layers(i.e., the denser spun fat, and the lightweight beads) will end uphaving to swap positions. The heavier cells will be driven through thelighter beads, toward the bottom end of the tube; and, the lighter beadswill be effectively trying to get out of the way of the heavier cells,and move toward the top of the tube while the heavier cells pass by ontheir way toward the bottom of the tube.

The net result is that the cells and the beads will bump into, pushagainst, and jostle each other, as they move in opposite(“counter-flow”) directions, in ways which will help pry the cells loosefrom the unwanted extra-cellular components of the spun fat.

Therefore, as above, this design (using bead-filled centrifuge tubes)merits optimization testing, to determine out whether this design canprovide cell viability levels in the vicinity of 85% (or possibly evenbetter), as achieved by the Inventor herein using extrusion devices.

Anyone contemplating this design should also note that beads of thistype can be manufactured in ways that likely can enhance their cellseparating-while-protecting capabilities; and, it should also be kept inmind that different types of beads, having different formulations,sizes, shapes, coatings, or other traits, can be mixed together, tocreate potentially even more effective mixtures. As two examples:

(1) some of the beads in a mixture can be made of aggressivelylipophilic (i.e., oil-adsorbing) material, comparable to the layer ofmaterial shown in FIG. 7, for sequestering oil from the cells, toprevent the oil from coating and suffocating the cells; and,

(2) some of the beads can be coated with antibodies that will bind tocollagen fibers, in ways which may be able to keep the collagen fiberscloser to the top of the tube, while the cells detach from the collagenand travel toward the bottom.

Accordingly, this type of “packed bed separation” may end up fittingnicely into the variety of “packed bed” arrangements and columns thatare commercially available, and which are widely used both in researchand in manufacturing, to separate numerous other types of biological orother chemical materials.

Additional Comments on Optimization Testing of Various Designs

Several additional comments and teachings merit attention, by anyone whois contemplating a series of optimization tests to determine whichparticular centrifuge tube design will perform most efficiently andoptimally, in gently prying cells loose from extra-cellular debris, in aspun fat preparation.

The most important such comment, for making such tests easier, faster,and less expensive, is that such testing can be performed, using livecells and entirely realistic conditions, by using either or both of:

(1) centrifuged “spun fat” cell suspensions obtained from humans vialiposuction that was carried out for weight loss purposes; and,

(2) processed and centrifuged cell-containing fatty tissues obtainedfrom cows, pigs, or other livestock that are processed at aslaughterhouse or rendering plant.

When human fat is removed via liposuction for weight loss purposes, thevolume typically is measured in liters, rather than milliliters.Therefore, a single weight-loss liposuction operation can providesufficient material to support a very large number of tests, to optimizethe exact style and dimensions of the extrusion cylinder and othercomponents (and operating parameters) for the devices described herein.

In addition, the performance of human stromal precursor cells, inmechanical separation and concentration systems, is likely to be modeledvery closely by (and, indeed, may be functionally identical to thebehavior of) cells from any type of large mammal, including cows, pigs,and other animals that are killed under controlled conditions inslaughterhouses. Accordingly, fatty tissues from livestock animals canprovide an abundant supply of stromal precursor cells, in liquefiedpreparations that can be created to emulate liposuction fluids.

The foregoing methods for treating cells have been able to createpreparations containing stromal precursor cells with substantiallylarger numbers of healthy and viable cells, than any prior treatmentsknown to the Applicant. The best prior techniques known to the Applicantgenerated cell populations with roughly 50% viability levels, asindicated by cell staining tests, using well-known staining reagents,such as a standard and widely-used mixture of both acridine orange (AO)and propidium iodide (PI), since that combination allows both viable andnon-viable cells to be counted, using high-speed flow cytometryequipment, This staining method (usually referred to as AO/PI staining),is well known, and is described in more detail in Example 4.

By contrast, the new techniques, which involve forcibly driving cellsthrough an extruder device having holes that are 0.5 mm in diameter, attemperatures of about 105° F., have been able to repeatedly achieve an85% viability level (i.e., using light microscopes and staining methods,estimates of viable cells within a microscopic field stand at roughly85%, with the proviso that that percentage number can be clarified andestablished with greater accuracy by using more advanced testingmethods, such as flow cytometry using large numbers of cells.

Accordingly, that 85% viability standard should now be regarded as abenchmark, against which other candidate techniques should be compared.

Now that this type of highly useful cell separation has been recognized,tested for efficacy, shown to work quite effectively, and therebyenabled by the teachings herein, any of the candidate designs disclosedherein can be tested, to determine whether any of them can reach (orpossibly even surpass) the 85% cell viability levels that have alreadybeen achieved by the Applicant herein.

It should be noted that numerous types of staining reagents, fluorescentbeads, and other materials that are taken in by viable cells, but not bynon-viable cells, are well known to researchers, including various typesof reagents that can be used in conjunction with automated equipment,such as flow cytometers, computerized analysis of photographic slides,etc. Any early-stage testing of stromal precursor cells, for viabilitylevels after a spun fat separation process as disclosed herein,preferably should use at least two different viability-assessingreagents which act by different biochemical mechanisms, to avoid anyartifacts or unforeseen problems that might arise if only a single typeof test is used to measure a crucial parameter.

Dimensions for 20-cc Syringes that Will Fit into Standard CentrifugeCartridges for PRP Machines

The Applicant herein uses a SMARTPReP™ centrifuge system, sold byHarvest Technologies for creating “platelet rich plasma” (PRP), so thedimensions discussed herein are based on measurements of that system. Itis believed that the relevant components of the MAGELLAN™ system havecomparable dimensions, and can be adapted accordingly, for use asdisclosed herein.

The SMARTPREP centrifuge has a single rotor, with two opposed andbalanced “arms” mounted on opposite sides of a vertical axle whichrotates at high speed. Each arm of the rotor holds a “cup” (which canalso be called a basket, holder, cartridge holder, or similar terms) atthe outer end of the rotor. The two cups are diametrically opposed toeach other, for proper balance and minimal vibration during high-speedrotation.

Each cup is mounted at one end of the rotor arm, by means of two pins onopposite sides of the cup. Those two pins, and accommodating supportmechanisms in the rotor arms, interact to form a rotatable support foreach cup. This allows the “bottom” of each cup (and the bottom of acartridge held by a cup, and the bottom of a syringe held by acartridge) to rotate outwardly, into an essentially horizontal positionduring high-speed rotation, due to centrifugal force. As mentionedabove, centrifugal forces should be limited to about 40 G, to avoiddamage to the cells.

The following discussion, concerning the dimensions of the cartridgesand cups in a PRP centrifuge, arises from the fact that, in mostphysicians' offices and clinics, for convenience and speed, it ispreferable to be able to use a single desktop centrifuge for both of twodifferent types of centrifugation steps (i.e., (i) for centrifugingblood, to obtain PRP; and, (ii) for centrifuging syringes that containfatty tissues and cells obtained by liposuction), without requiringsubstantial delays or alterations in the machine, between those twosteps.

However, it should be recognized that the standard rotor arm, in acentrifuge designed for PRP isolation, can be removed and replaced by adifferent rotor arm, relatively quickly and without requiring anyspecialized tools, merely by: (i) unscrewing and removing a specializedretainer cap from the top of the vertical axle which supports the rotorarm; (ii) lifting off and removing the standard rotor arm from thevertical axle; (iii) replacing the standard rotor arm with a differentrotor arm which can have a shorter length if desired; and, (iv)replacing the retainer cap on the axle, in a manner which secures thenew rotor arm to the axle.

Therefore, longer and deeper rotor cups can indeed be used, to hold andsupport longer and deeper cartridges that are designed to holdstandard-sized 20 cc syringes. This can be accomplished, fairly easily,merely by removing a standard rotor arm, and temporarily replacing itwith a shorter rotor arm which will allow a deeper cup to be usedwithout the bottom of the cup approaching too closely to the inner wallof the centrifuge chamber.

If it is decided to not use a shorter rotor arm, to enable the use ofdeeper cups and longer cartridges, then careful attention will need tobe paid to the dimensions of the cups, cartridges, and syringes thatwill be involved during centrifugation of a fluidized liposuctionextract.

The standard cups that normally are contained in a SMARTPReP centrifugehave internal diameters of about 3 inches (about 7.6 cm), and depths ofabout 3.4 inches (about 8.6 cm).

A widely used and standardized type of 20 cc syringe (made ofinexpensive plastic, and designed to be discarded after a single use, toavoid risks of contamination) has an internal diameter of 1.9 cm, and atotal length of 10 cm when the plunger handle has been removed. Becauseof the “headroom” that is available, which normally allows the cups andcartridges to swing into an outwardly horizontal position when the rotorbegins to spin, it is believed that those types of standardized 20 ccsyringes will be able to fit into specially-adapted centrifugecartridges that will fit into a SMARTPReP centrifuge. However, that willcreate a “tight fit”, which will constrain and limit the thicknesses(and therefore the strength and durability) of the “walls” and “floors”that are used to make such a centrifuge cartridge.

To provide a more “comfortable and convenient” system (rather than acrowded and compacted system that can barely fit into the availablespace), slightly deeper centrifuge cups can be provided, which canutilize some portion of: (i) about ¼ inch of “gap” space that exists,between the bottoms of the standard cups, and the inner wall of acentrifuge chamber, and/or (ii) about ½ inch of “headroom” space, in thearea where the centrifuge cups are mounted to the ends of the rotorarms.

Alternately or additionally, somewhat shorter syringes, with syringebarrels having internal diameters wider than the 1.9 cm which is used inthe standardized syringes described above, can be provided.

When accommodations are made for wall thicknesses (assuming at least 2mm wall thicknesses for centrifuge cartridges that will be reused, and 1mm wall thicknesses for syringes), there is sufficient room within astandard centrifuge cartridge (having an internal diameter of 3 inches,or about 7.6 cm) for three syringes, with each syringe having aninternal diameter of up to about 2.6 cm.

A standard 20 cc syringe with an internal diameter of 19 mm (radius=9.5mm) requires a calculated length of 7.035 cm to hold 20 cc of liquid;however, when the additional volume of liquid that will be contained inthe syringe tip is also included, the measured length, from the inside“shoulder” surface of the syringe to the 20 cc marking line on thebarrel, is only about 6.4 cm. The remaining 3.6 cm of syringe length isoccupied by the tapered tip (about 1.2 cm), and the opening or “throat”portion of the syringe (about 2.4 cm).

By contrast, if a 20 cc syringe were to be manufactured with an internaldiameter of 2.6 cm (radius=13 mm), it would require a calculated lengthof only 3.77 cm, rather than 7.035 cm as in a standard syringe, to hold20 cc of liquid. Accordingly, if the tip and throat lengths wereunchanged, a syringe with 2.6 cm internal diameter could be reduced, intotal length, from 10 cm for a standard syringe, to about 6.7 cm for themodified syringe.

However, a syringe that short, wide, and “stubby” likely would not feelnormal or “comfortable” in the hands of most physicians who performliposuction. Since liposuction is an invasive procedure, in which “feel”and tactile sensations play important roles in helping a surgeon orphysician remove fatty tissue without damaging surrounding tissue, ajump from 19 mm to 26 mm, in syringe diameter, would not be optimal.Instead, smaller increases in diameter, such as to about 20 to 22 mm ininternal diameter, are regarded as preferable. If a syringe has aninternal diameter of 21 mm, which is only 2 mm wider than a standard 20cc syringe, it will require only 5.26 cm of length to hold 20 cc,compared to 6.4 cm for a standard syringe. That reduction in length, ofnearly 1.4 centimeter compared to a standard 20 cc syringe, can provideample clearances in all dimensions and directions.

Similarly, an increase in internal diameter of a single millimeter, from19 mm (standard) to 20 mm (modified), could provide a reduction inoverall length of about 6.7 mm, compared to a standard 20 cc syringe.That relatively modest reduction in length likely would be sufficient toprovide a reasonable balance between “clearance” and “comfort”, forsyringes that will be short enough to fit into cartridges that will fitinto PRP centrifuges such as the SMARTPReP system, in a manner that willenable two centrifuge cartridges to hold a total of six 20 cc syringes(three syringes in each cartridge) in each “run”.

Accordingly, FIG. 13 is a perspective view of a centrifuge cartridge900, which is made of clear plastic (such as an acrylic, carbonate,etc.), and which is provided with three wells 902, 904, and 906, whichare sized to hold a 20 cc syringe in each well. The outer cylindricalwall 910 of cartridge 900 is also provided with a “notch” 912. Thisnotch is sized and designed to interact with an accommodating protrusion(often called a “key” or similar terms) in the inner wall of acentrifuge cup. The notch must be properly aligned with the key, inorder to insert the cartridge into the cup. This system, which isstandard in PRP centrifuges, helps prevent and minimize any vibration,rotation, rattling, or other unwanted motion of the cartridge, withinthe cup, during high-speed centrifugation.

FIG. 14 is an overhead (plan) view of a centrifuge rotor 950, with avertical axle 952 coupled to a motorized drive unit (not shown) havingrotor arms 960 and 970, which will rotate in a horizontal plane.Arc-shaped (semi-circular) support mechanisms 962 and 972 are positionedat the ends of rotor arms 960 and 970, and each support mechanism has apair of strong round-tip pins 964 and 974. These pins interact withaccommodating attachments in a strong cup 980 (usually made of metal),which is designed to accommodate plastic cartridges in a manner whichwill provide evenly-distributed support to the plastic cartridges. Tosimplify the drawing, it is assumed herein that each plastic cartridge900 has a “lip” around its periphery, which rests upon and is supportedby the rim of the cup; therefore, the only portion of each cup 980 whichis visible from above is the “key” portion of the cup, which fits intothe notch (shown by callout number 912, in FIG. 13) of a plasticcartridge 900.

The suspension system which mounts each cup 980 on twodiametrically-opposed pins 964 (at the end of rotor arm 960) or 974 (atthe end of rotor arm 960) creates an imaginary axle which passes througheach pair of pins. That “axle” arrangement allows the bottoms of cups980, and plastic cartridges 900 (carrying loaded syringe barrels), toswing outwardly, due to centrifugal force, so that they reach anessentially horizontal (i.e., radial) position, while the centrifugespins at high speed.

Any of several approaches can be used to ensure that syringes loadedwith liposuction fluid are placed in the centrifuge cartridges in abalanced and symmetric manner, regardless of the number of loadedsyringes that are involved. These options include labeling (by ink, orby raised, submerged, or etched letters, etc.) the tops of the plasticcartridges with information to establish the preferred loading sequence,as indicated by the remarks in quotes shown in FIG. 14, as follows:

(1) If only two syringes will be centrifuged (i.e., with one syringe ateach end of the rotor), they should each be loaded into a well that ismarked with a phrase such as “Solo or 3/3”. That well should bepositioned along the “centerline” of the rotor arm.

(2) If four syringes are being centrifuged, they should be loaded intothe four wells that are labeled as “1 of 2” and “2 of 2”, to maintainbalance and symmetry.

(3) If six syringes are being centrifuged, then they must and willoccupy all six wells.

If that loading sequence is used, balanced and symmetric weighting willbe sustained, regardless of whether two, four, or six syringes are beingcentrifuged. This will avoid placing any non-symmetric stresses on therotor, or on the supporting pins or other components. While the amountof unbalanced stresses that would be imposed on the support pins andcups (and on the machine), if a different loading sequence is used,would not be great, and would not significantly damage the machine in asingle session, the types of stresses arising from even a mildlyoff-balanced loading should not be imposed on a centrifuge machinerepeatedly (such as during multiple hundreds or thousands of differentusage sessions), because stresses due to unbalanced loading will causeincreased wear and degradation of various bearings, couplings, and othercomponents, over a span of years.

It also should be noted that balancing weights, such as syringe-typebarrels filled with water or any other fluid or other weights, can beused to offset and balance out any syringes that have been loaded into acentrifuge. These types of “counterweights” are conventional, and areroutinely used whenever a single loaded tube, vessel, or other container(or an odd number of loaded tubes or containers) would otherwise createunbalanced loading, during centrifugation.

Indeed, in order to ensure that closely-balanced weightings will usedfor each and every “run” in a centrifuge machine, it is deemed advisableand preferable to provide physicians' offices that will be using thissystem, with a small and convenient mechanical balance (which can alsobe called a “balance scale” or similar terms). This type of scale canhave a balance arm mounted on a centered pivoting device, having cups ateach end that are shaped identically to the cups in a centrifuge, andwith a needle or pointer in the middle of the balance arm, which willpoint vertically toward a “Proper balance” indicator when the balancearm is exactly horizontal. A plastic cartridge, pre-loaded with the fullcomplement of loaded syringes barrels that will be centrifuged withinthe next few minutes, is placed into each of the two cups in the balancescale. If that balancing operation indicates that the two fully-loadedcartridges have unbalanced weights, a small quantity of water can beadded to well 920 of the lower-weight cartridge, until the weights ofthe two loaded cartridges are exactly balanced. If and when the weightsof the two cartridges are in good balance, each cartridge (with thesyringes it is carrying) is simply lifted out of the balancing scale,and placed in one of the cups in the centrifuge machine.

As a final comment prior to the examples, a phrase used in certainclaims, below, needs to be clarified. That phrase is, “a concentratedpreparation of stromal precursor cells, having a substantially reducedquantity of extracellular material, compared to concentrated fattytissue extract that has not been processed by the steps listed herein.”As used and intended in that phrase, a “substantial reduction” isregarded, by the Applicant herein, as occurring if the amount (measuredby either volume, or weight) of extracellular collagen fibers, and/orextracellular fat, is reduced by about 25% or more. In the still-earlytests described herein, the levels of reduction that were seen were muchhigher than that. Nevertheless, a reduction of even just 25%, in thevolume and quantity of unwanted and unproductive materials that will beinjected into a patient (when compared to “spun fat” injections) is ahighly desirable result. Therefore, that number has been chosen for useherein as a “benchmark” level for interpreting the phrase, “asubstantially reduced quantity of extracellular material, compared toconcentrated fatty tissue extract that has not been processed by thesteps listed herein.”

EXAMPLES Example 1: Extraction of Fatty Tissue Via Liposuction

The patient will be sterilely prepped and draped. A skin wheal,typically on one side of the abdomen or in a thigh or buttocks area,will be raised, initially using a small and thin needle, such as a 27gauge (27G) needle, which can deliver a saline solution containing ananesthetic such as xylocaine if desired, or which can be used after atopical anesthetic (such as a benzocaine ointment) has been applied tothe skin in that area. After the initial wheal is raised using a verysmall needle, a larger needle (such as a 3″ 25G needle) can be used,with a fanning-style injection technique, to inject 5 cc of 1% xylocainethrough the subcutaneous fat. Once this has been done, an even largerneedle, such as an 18G needle, can be used if desired. When the finalsharp-tipped needle is being withdrawn from the anesthetized wheal, itssharp beveled tip is used to make a somewhat enlarged nick in the skin,to accommodate an injector cannula.

A saline/xylocaine mixture is prepared using 1000 cc of 0.9% sodiumchloride solution and 50 cc of xylocaine 1% with epinephrine. Of thatmixture, 20 cc will be spread through the wheal area, using a rigidinjector cannula coupled to a syringe. These injector cannulae are oftencalled “Tulip injectors”, because standard and preferred models are soldby a company called Tulip Products; their website, www.tulipmedical.com,describes and illustrates devices and accessories that are commonly usedduring liposuction procedures.

A fanning-style injection technique should generally be used todistribute the liquids under the skin, into subcutaneous fat. Thecannula should be kept generally tangential to the skin surface, so thatit penetrates only shallowly into the fatty layer and does not penetratethe underlying muscles or membranes. During the injection process,lateral motion of the Tulip injector tip (which releases fluid out ofthe cannula via orifices on the side of the tube, rather than directlyfrom the tip of the tube) is used to break up the fat, as fluid is beinginjected into and passed through the area. Generally, a semi-circulararea (rather than a completely round circle) is used for extraction;however, the physician can extract fluid from an area having any sizeand shape, depending on factors such as the surface contours of thepatient's body or limb in that region.

It should also be noted that, if desired, stromal precursor cells foruse in connective tissue repair as described herein can be obtained bymeans of a larger-volume liposuction procedure that will also serve aweight-reduction purpose. For example, if an overweight person is havingknee, hip, or ankle problems (as is fairly common), then a portion ofthe liquefied fatty tissue which is removed during a larger-scaleliposuction procedure (using general anesthesia, a relatively largeextraction cannula, etc.) can be processed as described herein, toisolate and concentrate a set of stromal precursor cells which can thenbe injected back into one or more joints or other areas of discomfort ordamage, such as into either or both of the knee or hip joints.

After a fluid injection stage has been completed, the injector cannulawill be withdrawn and replaced by a Tulip extractor. If desired, anextractor cannula can be connected to a machine which can exert either:(i) a steady suction force on the extractor tube; or, (ii) a variablelevel of suction, which can be controlled by the surgeon (or anassistant) by various means, such as a foot pedal. However, mostsurgeons prefer to have manual control over the level of suction, duringat least part of the procedure, so that they can feel (with their hands)what is happening during the fluid withdrawal. Accordingly, that type ofsuction force is created when the surgeon pulls on the handle of theplunger, which travels inside the barrel of the syringe. If desired, thesurgeon can also use a “Johnnie Lok” (illustrated atwww.tulipmedical.com), which is a specialized type of clip that willtemporarily affix the plunger handle to the syringe barrel, in a mannerwhich sustains a level of suction inside the syringe while the surgeon'shands are freed up to do something else.

The liquefied fatty tissue will be extracted using a fanning technique.Each time a syringe barrel (20 cc is a preferred and convenient size)approaches a point of being full, it can be detached from the Tulipextractor, moved out of the way, and replaced by an empty 20 cc syringe.The full syringe can be either set aside for a few minutes while itawaits processing, or a surgeon's assistant can immediately beginprocessing the fluidized fatty tissue inside the syringe.

This extracting process can be repeated until the desired amount of fathas been harvested, using any number of 20 cc syringes.

Example 2: First Centrifugation Step

When a desired number of 20 cc syringes have been filled with afluidized liposuction extract, they are placed into two holdingcartridges which are designed for use in the type of centrifuge machinethe surgeon is using. As described above, a preferred approach involvesscrewing a small “blind” cap onto the threaded luerlock tip of eachsyringe, before the syringe is placed in a centrifuge cartridge.

The syringe plungers, which were used to establish suction during theliposuction process, are disengaged from the syringe barrels, before theloaded syringes are centrifuged, by unscrewing the tips of the plungersfrom accommodating threads in plunger tip or stopper components, madefrom rubber or a flexible polymer. Each rubber or polymer stopper willremain in position, within a syringe barrel, during centrifugation, andwill act as a watertight cap on the liquid in the syringe, which willhelp maintain sterility of the contents inside the syringe.

After two cartridges have been loaded with the desired number ofsyringes, they preferably should be weighed or balanced against eachother, to ensure that they have approximately balanced weights. Wheneverappropriate, additional weight should be added to a cartridge whichweighs substantially less than the cartridge it is paired against.

Once the syringes have been emplaced in the cartridge, and after theplunger handles have been removed, the cartridge is placed in thecentrifuge, and is centrifuged at about 40 G, for 8 to 10 minutes.

When the centrifugation of the liposuction extract is complete, therewill be three main layers, which are relatively easy to distinguish,visually. In the “bottom” of the tube will be an aqueous layer, with asubstantial number of stromal stem cells; accordingly, that watery fluidwill be passed through a cell-retaining filter, to keep the cells in theliquid that will be retained and used, while the water is removed. Todislodge the cells from the surface of the filter, a “pulse” of a wateryliquid (such as Hanks balanced salt solution, or HBSS) will be forcedthrough the filter.

In the “top” of the tube will be an oily liquid, which will have few ifany viable cells. That layer will be discarded.

The center layer, referred to herein as a “spun fat” material willcontain the majority of the viable stromal stem cells. It will be savedfor subsequent processing and use.

In a number of early tests, the “spun fat” layer was mixed directly withplatelet-rich plasma (PRP), using the same 2:1 ratio described below,and injected back into the patient, into a connective tissue repairsite. The results of most of those tests were entirely acceptable, andthose treatments provided substantial and even major benefits to most ofthe patients who were treated with stromal stem cells from a “spun fat”layer after a single centrifugation step.

However, additional processing steps were subsequently developed, tofurther purify and concentrate stromal stem cells from a liposuctionextract. Those additional steps are now regarded as establishing thepreferred “best practice” mode for treating a liposuction extract toconcentrate stromal stem cells from the extract, before the stromal stemcells are reinjected back into a patient. Accordingly, those additionalprocessing steps are described in the next example.

Example 3: Collagenase Treatment and Second Centrifugation

The layer of “spun fat” from the first centrifugation step will containa substantial amount of extra-cellular debris, including glycogenparticles, and strands of collagen, the fibrous protein which createsthe extra-cellular matrix that holds cells together in any type of softtissue.

As described above, one method for breaking apart and removing theremnants of the extra-cellular collagen matrix (which otherwise cancause viable stem cells in the spun fat layer to clump together inundesirable ways) involves treating the “spun fat” from the firstcentrifugation step (supplemented by cells that were filtered out of thewatery layer that was formed during the first centrifugation), with anaqueous salt solution, and with collagenase, an enzyme that will digestand break apart collagen fibers.

As mentioned above, this type of cell-concentrating treatment, using anenzyme, is no longer preferred, since a better and faster mechanicalmethod was subsequently developed, as described above and in Example 4,below.

Because of the volumes involved, an enzymatic incubation step (if one isperformed) can be carried out in a different incubation chamber, whichshould be designed for placement directly into a centrifuge when thecollagenase incubation step is complete. Typically, a commerciallyavailable dry bovine collagenase A type 1 preparation (sold bySigma-Aldrich) is mixed with 25 cc of Hank's balanced salt solution(HBSS, sold by Gibco-BRL, and which should not contain phenol redindicator) to prepare a 0.2% (by weight) concentration of thecollagenase. 50 cc of “spun fat” with stromal stem cells is added to the25 cc collagenase solution, and the chamber is shaken vigorously for 5to 10 seconds. The mixture is then incubated for an hour at 37 o C, withadditional shaking every 10 to 15 minutes.

At the end of that incubation period, the 75 cc cell-and-collagenasemixture is diluted 9:1 (i.e., to a final 10% level), by adding 675 cc ofeither or both of the following: (i) commercially-available fetal bovineserum, and/or (ii) platelet-poor plasma, preferably from the samepatient who is being treated (about 25 cc of platelet-poor plasma willbe generated from a 60 cc aliquot of whole blood).

The resulting mixture is vigorously shaken for several seconds, toensure that any stromal stem cells in the collagenase-treated cellpreparation can be released from any remaining collagen fragments orother debris. It is then centrifuged at about 40 G for about 8 to 10minutes. The relatively dense stromal stem cells will settle into thebottom of the centrifuge chamber. If the bottom of the chamber is flat,the stromal stem cells will form a layer; if the bottom of the chamberis conical, the stromal stem cells will form a pellet. The supernatantis discarded, and the concentrated stromal stem cells, which will be ina relatively thick and viscous material, comparable to a paste, areready to be mixed with platelet-rich plasma (PRP).

Example 4: Cell Extrusion Driven by a Balloon Catheter

As a preferred alternative to an enzymatic incubation step usingcollagenase, a mechanical method and device were developed and tested,for gently dislodging and detaching stromal precursor cells from theextra-cellular collagen matrix that is normally present in thepost-centrifuge “spun fat” layer that is obtained from liposuctionedmaterial.

This mechanical approach is now strongly preferred over a collagenaseincubation step, for two crucial reasons.

First, mechanical separation can be performed much more rapidly, such aswithin 5 minutes or less, while an adequate collagenase incubation stepis likely to require at least an hour. Shorter processing periods arehighly advantageous, since the viability and health of the stromalprecursor cells that are injected back into the body of the patient isinversely related to the amount of time the cells spent outside thebody. Results seen by the Applicant have indicated that if connectivetissue cells that have been extracted from a body can be returned tothat same body within about 20 minutes or so, they do not seem to sufferfrom a serious drop in viability. By contrast, if the cells are keptoutside the body for more than an hour, a type of biochemical “shock”commences and then picks up steam, and will substantially reduce theirviability.

The second major factor involves regulations and governmentrequirements. For valid and appropriate reasons, regulatory oversightshifts into increased scrutiny and caution, if cells that have beenremoved from a body are treated by enzymes or other chemicals, beforethe cells are returned to the body. Questions will necessarily ariseover the preparation and purity of the enzymes or other chemicals, overany quantities of such enzymes or chemicals that might be present a cellpreparation when it is returned to the body, and over any short-termand/or long-term effects that the enzymatic or other chemical treatmentmight be inflicting on the cells. Those questions will need to beaddressed by careful testing.

By contrast, if the cell-concentrating treatments are entirelymechanical, and if the only result of those treatments is to furtherconcentrate the cells while reducing unwanted debris and contaminants inthe final preparation, a valid presumption arises that such mechanicaltreatments are safe, beneficial, and effective, and are notsubstantively different from centrifugation or other mechanicaltreatments. Accordingly, heightened government scrutiny (and theadditional expense of conducting clinical trials to prove safety) willnot kick in, unless and until adverse results indicate that additionalscrutiny should commence.

Accordingly, FIG. 6 depicts one of several types of mechanicalprocessing systems that were developed and tested by the Applicantherein, for gently forcing a “spun fat” preparation through 0.5 mmextrusion holes. The Applicant, a highly skilled surgeon who has beendoing precision work with his hands for decades, fashioned this assemblyout of available materials, using a standard centrifuge tube as theouter holding device, and a smaller cylinder of plastic, drilled with alarge number of holes, as the extrusion cylinder which was centered,using glued supports, inside the centrifuge tub. A deflated ballooncatheter was placed inside the extrusion cylinder, and the assembly wasmounted on top of a conventional electric heating surface, which wasadjusted to maintain the temperature of its contents within a range ofabout 103 to 105° F. That slightly elevated temperature range was foundto be highly beneficial, in helping promote cell dislodgement andconcentration without damaging cell viability.

A quantity of spun fat, from a liposuction procedure on a human patient,was loaded into the extrusion cylinder, around the balloon catheter. Thecatheter was then inflated, using a standard catheter inflator with amanually rotated displacement shaft inside an outer cylinder. Theexpansion of the balloon catheter forcibly pressed the spun fat materialout through the holes in the extrusion cylinder, into an annularcollection zone which occupied the remainder of the centrifuge tube. Theextruded material was then centrifuged to separate the cells (which werecollected) from the extracellular debris and fat (which were discarded).

The processed cells were then examined for viability, using a stainingreagent combination that contains both acridine orange (AO) andpropidium iodide (PI). This staining method (usually referred to asAO/PI staining) is well known, and is well-suited for use withhigh-speed flow cytometry equipment, since it allows both viable cells,and non-viable cells to be counted very rapidly. The AO reagent willenter all cells, whether viable or dead; by contrast, the PI reagentwill enter cells only if they are living and viable. As a result, livingcells will appear fluorescent green, from the PI reagent, while deadcells will appear fluorescent orange, due to the combination of both AOand PI.

Based on cell counts using that system, once the Applicant optimized andbecame comfortable with the steps, speed, and parameters of his system,he was able to repeatedly achieve viability levels of about 85%, amongthe cells that were processed by the method described above.

Furthermore, after stromal precursor cells had been processed andconcentrated by that device, they formed a liquid suspension which couldbe injected using relatively thin and small hypodermic needles, such as18 or even 20 gauge needles. By comparison, most injections of “spunfat” materials which has not had that type of extrusion processingrequire substantially larger-diameter needles, such as 12 or 14 gaugeneedles.

Example 5: Preparation of Platelet-Rich Plasma

To prepare a sufficient quantity of platelet-rich plasma (PRP),approximately 60 cc of blood is withdrawn from the patient. The bloodcan be processed directly in a SMARTPReP™ unit (sold by HarvestTechnologies), which is a specialized unit that will create about 5 to10 cc of PRP from 60 cc of whole blood. The blood should be processedpromptly after withdrawal, to isolate a platelet-rich plasma extractwhich will be injected back into the patient; if desired, aplatelet-poor plasma liquid can also be collected, and can be usedduring the second centrifugation step, as described above. Anyplatelet-rich or platelet-poor plasma preparations should be stored in amedical-grade freezer, at a suitable temperature, such as −80° C.

It should also be noted that 25 cc of platelet-poor plasma (from 60 ccof whole blood) can be passed through a “mini-heme” concentrator, whichwill remove most of the water, to create about 1 to 2 cc of an enrichedfluid that will contain various growth factors and other polypeptidesand proteins. If desired, this concentrated proteinaceous fluid can beadded to the PRP liquid, before the PRP is mixed with the stem cellpreparation.

The PRP preparation will be mixed with the stromal precursor cellpreparation, and the mixture will be injected (with real-time imagery asa guide, such as via ultrasonic imaging) into a site in a patient's bodyor limb which is in need of connective tissue repair.

Thus, there has been shown and described a set of interconnecteddevices, machines, and methods for: (1) using minimally-invasiveliposuction methods to extract stromal precursor cells from a patientwho needs connective tissue repair; (2) processing the liposuctionextract to create a highly concentrated preparation of stromal precursorcells, while eliminating the large majority of any extracellular fat anddebris; and, (3) reinjecting the highly concentrated stromal precursorcells back into the patient, along with platelet-rich plasma if desired,at a site where connective tissue is damaged or defective. Although thisinvention has been exemplified for purposes of illustration anddescription by reference to certain specific embodiments, it will beapparent to those skilled in the art that various modifications,alterations, and equivalents of the illustrated examples are possible.Any such changes which derive directly from the teachings herein, andwhich do not depart from the spirit and scope of the invention, aredeemed to be covered by this invention.

REFERENCES

-   Abuzeni, P. Z., et al, “Enhancement of Autologous Fat    Transplantation With Platelet Rich Plasma,” American Journal of    Cosmetic Surgery 18(2): 59 (2001)-   Pietrzak, W. S., et al, “Platelet rich plasma: biology and new    technology,” J Craniofac Surg. 16(6): 1043-54 (2005)-   Maniscalco, P., et al, “The Cascade membrane: a new PRP device for    tendon ruptures,” Acta Biomed. 79(3): 223-6 (2008)-   Hall, M. P., et al, “Platelet-rich plasma: current concepts and    application in sports medicine,” J Am Acad Orthop Surg. 17(10):    602-8 (2009)-   Gociman, B., et al, “Caption: a filtration-based platelet    concentration system,” Expert Rev Med Devices 6(6): 607-10 (2009)-   Lacci, K. M., et al, “Platelet-rich plasma: support for its use in    wound healing,” Yale J Biol Med. 83(1): 1-9 (2010)-   Lopez-Vidriero, E., et al, “The use of platelet-rich plasma in    arthroscopy and sports medicine: optimizing the healing    environment,” Arthroscopy 26(2): 269-78 (2010)

The invention claimed is:
 1. A method for creating a cell preparationfor treating connective tissue, the method comprising the followingsteps: a. removing a liquefied fatty tissue extract containing stromalprecursor cells, from a patient; b. centrifuging the liquefied fattytissue extract at a speed and for a duration which separates theliquefied fatty tissue extract into an aqueous layer, a layer containingviable cells, and an oily layer; c. mechanically processing the layercontaining viable cells in a device in a manner which causes viablecells to be released, the device comprising: (i) a generally cylindricalouter wall; (ii) an extrusion barrier with holes passing through it,mounted within said outer wall; (iii) means for loading spun fat into aninterior first zone within said extrusion barrier; (iv) means forimposing elevated pressure on said spun fat which has been loaded intosaid first zone; and, (v) an annular second zone, outside of saidextrusion barrier, suited for receiving and collecting extruded materialwhich has been forced under pressure through said extrusion barrier;and, d. centrifuging the viable cells in the annular second zone of thedevice at a suitable speed and duration to create a concentratedpreparation of stromal precursor cells, wherein the aqueous layer fromthe liquefied fatty tissue extract is passed through a cell filter whichretains cells while allowing aqueous liquid to pass through for removaland disposal, and wherein said cells which were filtered from theaqueous layer are added to the concentrated preparation of stromalprecursor cells.
 2. The method of claim 1 wherein the mechanicalprocessing generates shearing forces and is selected from the group ofprocessing methods consisting of extrusion under pressure, screenpassaging, and shearing-force stirring.
 3. The method of claim 1 whereinthe mechanical processing generates shearing forces that will causeviable cells to be released from extracellular collagen fibers and isperformed at one or more temperatures within a range of about 103 toabout 115 degrees Fahrenheit.
 4. The method of claim 1 whereinplatelet-rich plasma is mixed with said concentrated preparation ofstromal precursor cells.
 5. The method of claim 1 wherein the layercontaining viable cells is treated with collagenase enzyme, for asufficient period to partially degrade extracellular collagen fibers inthe concentrated fatty tissue extract, thereby promoting release ofviable cells from degraded collagen fibers.
 6. A method comprising:introducing a fatty tissue extract to a cavity of an inner body of aconcentrator, the concentrator comprising: an outer shell; the innerbody positioned within and operatively coupled to the outer shell, theinner body comprising a plurality of holes and the cavity, wherein anannular gap is formed between the outer shell and the body; an extrusiondevice positioned in communication with the cavity, wherein theextrusion device is configured to mechanically force the fatty tissueextract through the holes and the extrusion device comprises a piston, aplunger, a balloon catheter, or a combination thereof; and mechanicallypressing the fatty tissue extract through the holes into the annular gapto release viable cells from the fatty tissue extract; and centrifugingthe viable cells in the annular gap of the concentrator to create aconcentrated preparation of stromal precursor cells.
 7. The method ofclaim 6, further comprising: centrifuging a liquefied fatty tissue toseparate the liquefied fatty tissue into an aqueous layer, the fattytissue extract, and an oily layer.
 8. The method of claim 7, furthercomprising: removing the liquefied fatty tissue extract containingstromal precursor cells from a patient.
 9. The method of claim 6,wherein the extrusion device comprises the balloon catheter andmechanically pressing comprises inflating the balloon catheter.
 10. Themethod of claim 9, wherein the balloon catheter comprises a stiffeningring operatively coupled to the inner body by a plurality of radialstruts.
 11. The method of claim 6, wherein the concentrator comprises anoutlet in fluid communication with the annular gap and centrifuging theviable cells in the annular gap of the concentrator to create theconcentrated preparation of stromal precursor cells positions theconcentrator preparation of stromal precursor cells adjacent to theoutlet.
 12. The method of claim 11, further comprising suctioning thestromal precursor cells out of the annular gap through the outlet. 13.The method of claim 11, wherein the concentrator comprises an inlet influid communication with the cavity of the inner body and the inlet isposition on a first end of the concentrator and the outlet is positionon a second end of the concentrator, the first end is oppositelydisposed from the second end.
 14. The method of claim 10, wherein theconcentrator comprises a distributor plate intermediate the inlet andthe cavity of the inner body.
 15. The method of claim 6, wherein theinner body comprises a range of 50 to 500 holes and the holes comprise adiameter of 0.5 mm.
 16. The method of claim 6, wherein the concentratorcomprises an oil sequestering ring positioned within the annular gap andfurther comprising separating oil, liquefied fat, or combinationsthereof from the concentration of stromal precursor cells utilizing theoil sequestering ring.
 17. The method of claim 6, wherein centrifugingthe viable cells in the annular gap of the concentrator to create aconcentrated preparation of stromal precursor cells is performed in aconventional desktop centrifuge machine.
 18. The method of claim 6,further comprising after mechanically pressing the fatty tissue extractand prior to centrifuging the viable cells in the annular gap:introducing a second fatty tissue extract to the cavity of the innerbody of the concentrator; and mechanically pressing the second fattytissue extract through the holes into the annular gap to release viablecells from the second fatty tissue extract.
 19. A method comprising:introducing a fatty tissue extract to a cavity of an inner body of aconcentrator, the concentrator comprising: an outer shell; the innerbody positioned within and operatively coupled to the outer shell, theinner body comprising a plurality of holes and the cavity, wherein anannular gap is formed between the outer shell and the body and whereinthe inner body comprises a range of 50 to 500 holes and the holescomprise a diameter of 0.5 mm; an extrusion device positioned incommunication with the cavity, wherein the extrusion device isconfigured to mechanically force the fatty tissue extract through theholes; and mechanically pressing the fatty tissue extract through theholes into the annular gap to release viable cells from the fatty tissueextract; and centrifuging the viable cells in the annular gap of theconcentrator to create a concentrated preparation of stromal precursorcells.
 20. A method comprising: introducing a fatty tissue extract to acavity of an inner body of a concentrator, the concentrator comprising:an outer shell; the inner body positioned within and operatively coupledto the outer shell, the inner body comprising a plurality of holes andthe cavity, wherein an annular gap is formed between the outer shell andthe body; and an extrusion device positioned in communication with thecavity, wherein the extrusion device is configured to mechanically forcethe fatty tissue extract through the holes; an oil sequestering ringpositioned within the annular gap and mechanically pressing the fattytissue extract through the holes into the annular gap to release viablecells from the fatty tissue extract; centrifuging the viable cells inthe annular gap of the concentrator to create a concentrated preparationof stromal precursor cells; and separating oil, liquefied fat, orcombinations thereof from the concentration of stromal precursor cellsutilizing the oil sequestering ring.